Compositions for use in treating or diagnosing bone disorders and/or cardiovascular disorders

ABSTRACT

The invention relates to methods for diagnosing bone disorders and/or cardiovascular disorders in a subject, by contacting a biological sample from the subject with (a) a nucleic acid molecule which hybridizes to miR-31 or its 5 or 3′ isoforms or variants, or (b) an agent that binds to miR-31 or its 5′ or 3′ isoforms or variants; detecting and evaluating the hybridization signal of the nucleic acid molecule or agent with a polynucleotide or said miR-31 or its 5′ or 3′ isoforms or variants; and comparing the detected and evaluated hybridization or binding signal with that of a control sample, wherein a stronger hybridization signal or a stronger binding signal in the sample of the subject compared to that of the control sample is indicative for a risk of developing or having a bone disorder and/or cardiovascular disorder

The present invention relates to compositions comprising anantagonist/inhibitor of a polynucleotide, said polynucleotide to beinhibited being capable of decreasing or suppressing expression of FZD3(Frizzled-3) or a biologically active derivative thereof for use intreating or preventing bone disorders and/or cardiovascular disorders.Such bone disorders comprise, inter alia, osteoporosis, osteopenia, bonefracture, bone cancer, as well as impaired bone homeostasis.Cardiovascular diseases to be treated by the compounds of the presentinvention may be selected from the group consisting of infarction,stroke, hypertension, thrombosis, vascular stenosis, coronary syndromes,vascular dementia, heart failure, renal failure, stress-relatedcarciovascular disorders, and atherosclerosis. Preferred compounds to beused in these medical interventions are antagonistic compounds, likenucleic acid molecules, directed to polynucleotides that are capable ofdecreasing or suppressing expression of FZD3 or a biologically activederivative thereof. An example of such a polynucleotide that needs to beantagonized is miR-31 or its 5′ or 3′ isoforms or variants. Also, thepresent invention relates to methods for and compositions for use indiagnosing bone disorders and/or cardiovascular disorders. Compounds tobe used in these diagnostic methods may be compounds (like primers andprobes) that are capable of detecting such a polynucleotide that iscapable of decreasing or suppressing expression of FZD3 or abiologically active derivative thereof. miR-31 (or miR-31 or its 5′ or3′ isoforms or variants) is, in accordance with this invention, apolynucleotide that is capable of decreasing or suppressing expressionof FZD3.

Accumulation of damage in cells and tissues has been accepted as one ofthe major driving forces of aging and age related diseases (Kirkwood,Cell (2005), 120: 437-474). One of the repair systems on tissue levelthat counteracts this functional decline are adult stem and progenitorcells. Their ability to self-renew and differentiate is essential forhomeostasis of tissues and organs. As adult human stem and progenitorcells with high differentiation potential have been identified indifferent tissues of the human body they would represent a pool of cellsthat should maintain high levels of tissue functionality. However, theirfunction also declines with age (Rando, Nature (2006), 441: 1080-1086).The old systemic environment has been identified to contain factors thateither fail to promote or actively inhibit successful tissueregeneration when tested in a parabiosis mouse model (Conboy, Nature(2005), 433: 760-764), while factors contained in the systemicenvironment of young animals promote successful tissue regeneration(Matsumoto, Eur Heart J (2009), 30: 346-355).

Visceral fat accretion is among the hallmarks of aging in humans(Huffman, Biochem Biophys Acta (2009), 1790: 1117-1123), whileosteogenic differentiation potential of ASCs decreases with age. Thisdecrease is not due to a loss of osteogenic precursors (Zhu, J TissueEng Regen Med (2009), 3: 290-301), suggesting that factors altering thecellular behaviour are involved. Furthermore, ASCs and endothelial cellsare linked in vivo since preadipocytes within adipose tissue depots andendothelial cells exhibit a close relationships (Hausman, J Anim Sci(2004), 82: 925-934), supporting a paracrine relationship between thesecell types.

However, the source of these factors altering the cellular behaviour wasunknown so far and only scarce knowledge is available on the identity ofthese factors, where Wnt and TGF-β signalling seem to be involved(Carlson, Aging Cell (2009), 8: 676-689). Besides various glands, onesource of secretion into the blood stream are endothelial cells per seand during older age, senescent endothelial cells, since they accumulateduring aging in vivo, especially at sites of atherosclerosis(Erusalimsky, Handb Exp Pharmacol (2006), 213-248; Erusalimsky, ExpPhysiol (2009), 94: 299-304); Minamino, Circ Res (2007), 100: 15-26).Several proteins that increase with senescence (Chang, Exp Cell Res(2005), 309: 121-136) have been identified, among them interleukin 8that is found at up to 50-fold higher levels in the supernatants ofsenescent endothelial cells (Hampel, Exp Gerontol (2006), 41: 474-481).

One of the transport mechanisms in blood has been described: variousfactors are packaged into exosomes, membrane-coated particles that aresecreted via exocytosis and play a role in cell-cell or organ-organcommunication by fusion with cells of target tissues (Caby, Int Immunol(2005), 17: 879-887). Exosomes, 40-100 nm in size, are membrane vesiclesof endosomal origin that are released into the extracellularenvironment. They can act on intercellular communication by allowingexchange of proteins, lipids, and also mRNA and miRNAs between cells(Valadi, Nat Cell Biol (2007), 9: 654-659; Viaud, PLoS One (2009), 4:e4942). The contained factors like miRNAs, mRNAs and proteins theninfluence the behaviour of the target cells. Examples are endothelialprogenitor cell derived exosomes that induce angiogenesis in endothelialcells upon uptake (Deregibus, Blood (2007), 110: 2440-2448),endothelial-derived exosomes in patients with pulmonary arterialhypertension (Bakouboula, Am J Respir Crit Care Med (2008), 177:536-543), ovarian carcinoma and glioblasotoma cells that releaseexosomes and change the tissue microenvironment in favour of tumorprogression (Keller, Cancer Lett (2009), 278: 73-81; Skog, Nat Cell Biol(2008), 10: 1470-1476). Especially exosomes released from tumour cells,which carry antigenic molecules recongnized by T cells, has suggested asa cell free antigen source for anticancer vaccines (Escudier, J TranslMed (2005), 3: 10; Iero, Cell Death Differ (2008), 15: 80-88; Morse, JTransl Med (2005), 3: 9; Viaud, Horm Metab Res (2008), 40: 82-88; Viaud,PLoS One (2009), 4: e49429).

However, mechanisms and factors involved in tissue regeneration or ininhibiting the same are still poorly understood and influencing theimpact of such factors on tissue regeneration is hardly possible.

This technical problem has been solved by the embodiments providedherein and the solutions provided in the claims.

Accordingly, the present invention provides for a composition comprisingan inhibitor of a polynucleotide, said polynucleotide to be inhibitedbeing capable of decreasing or suppressing expression of frizzled 3(FZD3) or a biologically active derivative thereof for use in treatingor preventing bone disorders and/or cardiovascular disorders in asubject as will be further detailed and exemplified herein below. Also,the present invention provides for a method for treating or preventingbone disorders and/or cardiovascular disorders in a subject comprisingadministering an effective amount of a composition comprising aninhibitor of a polynucleotide, said polynucleotide to be inhibited beingcapable of decreasing or suppressing expression of FZD3 or abiologically active derivative thereof. A polynucleotide to be inhibitedin context of this medical intervention (i.e. the herein disclosedmedical/pharmaceutical uses) and the methods of treating/preventing adisorder as provided herein may be miR-31 or its 5′ or 3′ isoforms orvariants. Furthermore, the present invention provides for a compositionfor use in and a method for diagnosing bone disorders and/orcardiovascular disorders in a subject. In context of the presentinvention, examples for such bone disorders are osteoporosis,osteopenia, bone fracture, impaired bone homeostasis or. Examples forsuch cardiovascular disorders are cardiovascular diseases such asstroke, infarction, hypertension, thrombosis, vascular stenosis,coronary syndromes, vascular dementia, heart failure and renal failure,as well as atherosclerosis (Erusalimsky, J Appl Physiol (2009), 106:326-32). As described in the appended examples, it could alsosurprisingly be shown that miR-31 is specifically elevated instress-induced senescent endothelial cells as well as exosomes ofstress-induced premature senescent endothelial cells. Accordingly, anantagonist/inhibitor of miR-31 is also to be used in context of thisinvention for the medical intervention of stress-related cardiovasculardisorders, like cardiovascular disorders due to oxidative stress orhypoxia/reperfusion damage.

In accordance with the above and the experimental data provided herein,the present invention relates to a composition comprising anantagonist/inhibitor of a polynucleotide that is capable of decreasingor suppressing expression of FZD3 or a biologically active derivativethereof and/or an antagonist/inhibitor of miR-31 or its 5′ or 3′isoforms or variants for use in treating or preventing bone disordersand/or cardiovascular disorders in a subject. Preferably, said subjectis a human subject. Also provided is a method for treating or preventingbone disorders and/or cardiovascular disorders in a subject, said methodcomprising administering an effective amount of a composition comprisingan antagonist/inhibitor of a polynucleotide that is capable ofdecreasing or suppressing expression of FZD3 or a biologically activederivative thereof and/or administering an effective amount of acomposition comprising an antagonist/inhibitor of miR-31 or its 5′ or 3′isoforms or variants. Again, also in this method of treatment the mostpreferred subject to be treated is a human subject in need of medicalintervention.

In context of the present invention, it has surprisingly been found howthe senescent secretome and especially senescent cell derived exosomesinfluence adult stem cells and, thus, tissue regeneration. For thispurpose, the present inventors investigated two prime characteristics ofsuch stem cells: self renewal/cell division as well as differentiationpotential. As models for investigating a possible effect of thesenescent endothelial secretome on adult stem cells, adipose-tissuederived stem cells (ASCs) were selected.

Furthermore, in context of the present invention it was surprisinglyfound that senescent endothelial cells secrete miRNAs packaged intoexosomes, that these vesicles are taken up by target cells suggesting aparacrine signalling function and that the amount of specific miRNAsdiffers in young versus senescent endothelial cells in vitro. As hasbeen found in context of the present invention, a microRNA (miRNA),miR-31, is markedly increased in the supernatant of senescent cells,protected and transported by exosomes, as well as in the blood of asubgroup of elderly donors. Furthermore, in context of the presentinvention, it has been found that miR-31 is taken up by ASCs by exposingthem to supernatant or purified exosomes of senescent endothelial cellsas well as by exposure to blood-derived exosomes of elderly individuals.

In context with the present invention, the target of miR-31 that isresponsible for osteogenic inhibition was identified as frizzled-3(FZD3), which so far has only been described as important for thedevelopment of the neuronal system since FZD3 knock-out mice showdefects in aconal growth and guidance (Endo, Mol Cell Biol (2008), 28:2368-2379; Stuebner, Dev Dyn (2010), 239: 246-260; Wang, J Neurosci(2006), 26: 2147-2156; Wang, J Neurosci (2002), 22: 8563-8573; Wang, JNeurosci (2006), 26: 366-364). Furthermore, it has been found hereinthat miR-31 inhibits osteogenic differentiation and increasesproliferation of ASCs. These surprising findings show thatpolynucleotides decreasing or suppressing expression of FZD3, like (butnot limited to) miR-31, represent a novel marker of biological age or ofage-associated diseases like osteoporosis, osteopenia, bone fracture orimpaired bone homeostasis or cardiovascular diseases such as stroke,infarction, hypertension, thrombosis, vascular stenosis, coronarysyndromes, vascular dementia, heart failure and renal failure oratherosclerosis and the like. Furthermore, antagonists/inhibitors ofpolynucleotides that decrease or suppress the expression of FZD3, likeantagonists/inhibitors of miR-31 and/or of miR-31 or its 5′ or 3′isoforms or variants represents novel therapeutics. Accordingly, inparticular miR-31 and/or of miR-31 or its 5′ or 3′ isoforms or variantsrepresent novel therapeutic targets in all diseases that requireosteogenesis and bone formation, such as osteoporosis, osteopenia, bonefracture, impaired bone homeostasis, bone cancer, etc. as well as incardiovascular diseases such as stroke, infarction, hypertension,thrombosis, vascular stenosis, coronary syndromes, vascular dementia,heart and renal failure or atherosclerosis. Examples of 3′ and 5′isoforms of miR-31 are shown in Table 3.

In context of the present invention, the functionality of miR-31delivery was tested on the proliferation and differentiation capacity ofASCs. As has been found in context of the present invention, the cellnumbers reached within a batch were higher, while osteogenicdifferentiation was partially inhibited by senescent exosomes or miR-31alone.

Furthermore, in context of the present invention it was surprisinglyfound that miR-31 is upregulated significantly in sera of elderlydonors. This shows that exosomal delivery of miR-31 is also found in theblood and not only in vitro during cellular senescence. Furthermore, incontext of the present invention, using elderly blood-derived exosomes,inhibition of osteogenesis was again observed. So far, miR-31 was notdescribed to be present in serum derived exosomes. Accordingly, incontext of the present invention, miR-31 is a valuable tool as abiomarker for aging and age-associated such as osteoporosis, osteopenia,bone fracture or impaired bone homeostasis or cardiovascular diseasessuch as stroke, infarction, hypertension, thrombosis, vascular stenosis,coronary syndromes, vascular dementia, heart and renal failure oratherosclerosis.

Additionally, in context of the present invention, besides theupregulation of miR-31 in elderly subjects, miR-31 was found to beelevated in a first set of experiments in 2 out of 4 osteopeniapatients. In more detailed experiments as provided in the enclosedexamples, 7 out of 10 osteopenia patients either show a stable diseaseor progression to osteoporosis where miR-31 was found in blood serum.Furthermore, as shown in the appended examples, inhibition of miR-31improves osteogenic differentiation, while transient increase of miR-31results in decreased osteogenic differentiation. This findingdemonstrates that inhibition of miR-31 improves osteoblast formation.This is very useful in the medical invention of bone disorders, e.g.,osteoporosis. Furthermore, expression of miR-31 is indicative for suchbone disorders (and also for cardiovascular disorders). Therefore,specific assays for the detection of miR-31 are also provided herein.Such detection assays are preferably carried out on biological samples,like serum and blood plasma and the like. Osteoporosis is defined as adisease characterized by low bone mass and structural deterioration ofbone tissue, leading to bone fragility and an increased susceptibilityto fractures. Osteoporosis is an age-related systemic condition thatnaturally occurs among mammals, mainly in humans (Xu, Endocr Rev (2010),31(4): 447-505). Accordingly, the present invention also provides for agood and reliable biomarker for bone disorders, like osteoporosis and/orosteopenia. The biomarker provided herein is also useful in thediagnosis of cardiovascular disorders. The appended examples also showthat miR-31 is elevated in stress-induced senescent endothelial cells.Furthermore, as shown herein, miR-31 is elevated in exosomes ofstress-induced premature senescent endothelial cells. Accordingly,miR-31 is also indicative for cardiovascular disorders.

Generally, in accordance with the present invention, when referring to apolynucleotide to be inhibited in context of the present invention, saidpolynucleotide is capable of decreasing or suppressing expression ofFZD3 or a biologically active derivative thereof as described andexemplified in detail herein.

As could be demonstrated in the present invention, the expression ofFZD3 can be decreased or suppressed by polynucleotides described hereincontained in senescent exosomes, e.g., by hybridizing to the mRNA ofFZD3. Thereby, for instance, degradation of or prevention of translationof FZD3 mRNA can be induced, both resulting in suppression or decreasingof expression of FZD3. Accordingly, inhibition of the polynucleotidesdescribed herein which inhibit or suppress expression of FZD3 wouldincrease of expression of FZD3. In context of the present invention,ASCs undergoing osteogenic differentiation were tested for FZD3transcription. Indeed, it was upregulated after 4 days compared to cellstreated with control medium (FIG. 7A). Moreover, FZD3 levels weresignificantly downregulated after treatment with senescent exosomescompared to treatment with young exosomes and control treated cells(FIG. 7B). 24 h after miR-31 transfection, FDZ3 was also downregulated,but did not reach significant levels, which might be explained due tothe very low mRNA levels of FDZ3 when cells are not differentiating(FIG. 7C). Thus, FZD3 represents not only a marker but also a necessaryfactor for osteogenic differentiation and turns out to be a directtarget of polynucleotides to be inhibited in context of the presentinvention also in ASCs.

As described and exemplified in the present invention, inhibition of thepolynucleotides to be inhibited in context of the present invention isparticularly useful in the treatment or prevention of osteoporosis,osteopenia, bone fracture or impaired bone homeostasis or cardiovasculardiseases such as stroke, infarction, hypertension, thrombosis, vascularstenosis, coronary syndromes, vascular dementia, heart and renal failureor atherosclerosis in a subject.

In one embodiment of the present invention, the polynucleotide to beinhibited in context of the present invention, i.e. which is capable ofdecreasing or suppressing expression of FZD3 or a biologically activederivative thereof (such as miR-31), may be a microRNA (also abbreviatedherein as miRNA or miR) or a precursor thereof, a mimic microRNA or aprecursor thereof, an siRNA or a precursor thereof, a long non-codingRNA or a precursor thereof, an snRNA (small/short hairpin RNA) or aprecursor thereof, an stRNA (small temporal RNA) or a precursor thereof,an fRNA (functional RNA) or a precursor thereof, an snRNA (small nuclearRNA) or a precursor thereof, a snoRNA (small nucleolar RNA) or aprecursor thereof, a piRNA (piwi-interacting RNA) or a precursorthereof, a tasiRNA (trans-acting small/short interfering RNA) or aprecursor thereof, an aRNA (antisense RNA) or a precursor thereof, or asmall non-coding RNA or a precursor thereof. In accordance with thepresent invention, as artificial polynucleotides mentioned hereinabovemay have the same effect on the expression of FZD3 or biologicallyactive derivatives thereof (i.e. decreasing or suppressing saidexpression) as physiological polynucleotides mentioned hereinabove,inhibitors of such artificial polynucleotides may at the same time alsobe inhibitors of such physiological polynucleotides. As used herein,“precursors” of a polynucleotide to be inhibited in context of thepresent invention, i.e. which is capable of decreasing or suppressingexpression of FZD3 or a biologically active derivative thereof, may beforms of the respective polynucleotides as they occur during maturationof the respective polynucleotides. For example, in context of thepresent invention, precursors of a microRNA or a mimic microRNA may beprimary miRNAs (pri-miRNAs) or precursor miRNAs (pre-miRNAs) asoccurring during maturation of miRNAs. Both are single transcripts (i.e.ssRNA) that fold into a characteristic intramolecular secondarystructure, the so-called “hairpin loop”, which contains a stretch ofabout 18 to 23 base pairs, which may be interrupted by mismatches. Incontext of the present invention, precursors of siRNAs may be long dsRNAmolecules or shorter “hairpin loop” ssRNA molecules. Both types of thesesiRNA precursors may contain a stretch of base pairs without anymismatch. The current model for maturation of mammalian miRNAs isnuclear cleavage of the primary miRNA (pri-miRNA) which liberates a60-70 nt stem loop intermediate, known as the direct miRNA precursor orpre-miRNA. The mature about 18-23 nt long miRNA is yielded from one armof the stem loop precursor (Bartel, Cell (2004), 116: 281-297; Lee, EMBOJ (2002), 21: 4663-4670; Zeng and Cullen, RNA (2003), 9: 112-123). In apreferred embodiment of the present invention, the polynucleotide to beinhibited in accordance with the present invention is a microRNA or aprecursor thereof or a mimic microRNA or a precursor thereof. Thepolynucleotides described in and to inhibited in context of the presentinvention may be of any length. Preferably, the polynucleotide is about15 to about 100 nucleotides in length, more preferably about 18 to about27 nucleotides and most preferably about 20 to about 24 nucleotides.

In a specific embodiment of the present invention, the polynucleotide tobe antagonized/inhibited in context of the present invention, i.e. thepolynucleotide being capable of decreasing or suppressing expression ofFZD3 or a biologically active derivative thereof and/or the miR-31miR-31 or its 5′ or 3′ isoforms or variants to be antagonized/inhibited,may be selected from the group consisting of:

-   -   (a) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO: 1 (i.e. miR-31);    -   (b) a polynucleotide which is at least 80% identical to the        polynucleotide of (a);    -   (c) a polynucleotide comprising the nucleotide sequence of SEQ        ID NO: 2 (i.e. the seed sequence of miR-31: GGCAAGAU); and    -   (d) a polynucleotide according to (b) comprising the nucleotide        sequence of SEQ ID NO: 2 (i.e. the seed sequence of miR-31:        GGCAAGAU).

According to the present invention, identity levels of polynucleotidesrefer to the entire length of the nucleotide sequence of the referred toSEQ ID NOs. and is assessed pair-wise, wherein each gap is to be countedas one mismatch. For example, the term “identity” may be used herein inthe context of a polynucleotide to be inhibited in context of thepresent invention which has a nucleic acid sequence with an identity ofat least 80%, 85%, 90%, 95%, 97%, 98% or 99% to a polynucleotidecomprising or consisting of the nucleotide sequence of any one of SEQ IDNO: 1 (mature miR-31), SEQ ID NO: 2 (seed sequence of miR-31), or SEQ IDNO: 3 (pre-miR-31) as also shown in Table 1 herein, preferably over theentire length. Furthermore, in the context of the present invention, apolynucleotide to be inhibited in context of the present invention mayalso have a nucleic acid sequence with an identity of at least 80%, 85%,90%, 95%, 97%, 98% or 99% to a polynucleotide comprising or consistingof a nucleotide sequence consisting of the sequence of SEQ ID NO: 1 orSEQ ID NO: 3 as shown in Table 1 herein including one, two or morenucleotide(s) of the corresponding mature- or pre-miRNA sequence at the5′-end and/or the 3′-end of the respective seed sequence. For example,in the context of the present invention, a polynucleotide to beinhibited in context of the present invention may have a nucleic acidsequence with an identity of at least 80%, 85%, 90%, 95%, 97%, 98% or99% to a polynucleotide comprising or consisting of the nucleotidesequence AGGCAAGAUGC (i.e. the seed sequence of SEQ ID NO: 1 includingone nucleotide of the corresponding mature sequence at the 5′-end andone nucleotide of the corresponding mature sequence at the 3′-end). Iftwo nucleic acid sequences being compared by sequence comparisons differin identity, then the term “identity” refers to the shorter sequence andto the part of the longer sequence that matches said shorter sequence.Therefore, when the sequences which are compared do not have the samelength, the degree of identity preferably either refers to thepercentage of nucleotide residues in the shorter sequence which areidentical to consecutive nucleotide residues contained in the longersequence or to the percentage of consecutive nucleotides contained inthe longer sequence which are identical to the nucleotide sequence ofthe shorter sequence. Of course, as described above, a gap as “part ofconsecutive nucleotides” is to be counted as a mismatch. In thiscontext, the skilled person is readily in the position to determine thatpart of a longer sequence that “matches” the shorter sequence. Also,these definitions for sequence comparisons (e.g., establishment of“identity” values) are to be applied for all sequences described anddisclosed herein.

TABLE 1 miRNAs, miRBase ID (miRBase: http://www.mirbase.org, version number 15: released April 2010), andmature and pre-miR sequences (seed sequences underscored). SEQ miRBaseID miRNA ID Sequence NO. mature miR-31 MI0000089 AGGCAAGAUGCUGGCAUAGCU 1seed miR-31 MI0000089 GGCAAGAU 2 pre-miR-31 MI0000089GGAGAGGAGGCAAGAUGCUGG 3 CAUAGCUGUUGAACUGGGAAC CUGCUAUGCCAACAUAUUGCCAUCUUUCC

TABLE 2 Examples for nucleic acid molecules that a capableof hybridizing to the above identified miRNAs,seed sequences and the like. miRNA Hybidizing Sequence SEQ ID NO.mature miR-31 AGCUAUGCCAGCAUCUUGCCU 6 seed miR-31 AUCUUGCC 7 pre-miR-31GGAAAGAUGGCAAUAUGUUGGCAUAG 8 CAGGUUCCCAGUUCAACAGCUAUGCCAGCAUCUUGCCUCCUCUCC As used herein, thymine (T) and uracil (U) may beused interchangeably depending on the respective type of polynucleotide.

Such hybridizing sequences (or functional fragments or isoforms thereof)may be employed as specific antagonists/inhibitors polynucleotide thatis capable of decreasing or suppressing expression of FZD3 or abiologically active derivative thereof, and/or as specificantagonists/inhibitors an antagonist/inhibitor of miR-31 or its 5′ or 3′isoforms or variants. Such hybridizing sequences (or functionalfragments or isoforms thereof) may hybridize to all kinds ofpolynucleotides to be antagonized or inhibited as described herein,including microRNAs, siRNAs, mimic microRNAs, long non-coding RNAs,snRNAs, stRNAs, (RNAs, snRNAs, snoRNAs, piRNAs, tasiRNAs, aRNAs as wellas precursors of such RNAs.

TABLE 3 Examples of 5′ and 3′ isoforms of miR-31 SEQ SEQ ID ID5′-isoform NO. 3′-isoform NO. GAGGCAAGAUGCUGGCAUAG 9UGCUAUGCCAACAUAUUGCCA 23 CU UC AGGCAAGAUGCUGGCAUAGC 10UGCUAUGCCAACAUAUUGCCA 24 UG U AGGCAAGAUGCUGGCAUAGC 11UGCUAUGCCAACAUAUUGCCA 25 UGU AGGCAAGAUGCUGGCAUAGC 12UGCUAUGCCAACAUAUUGCCA 26 U UCU AGGCAAGAUGCUGGCAUAGC 13GCUAUGCCAACAUAUUGCCAU 27 C AGGCAAGAUGCUGGCAUAG 14 CUAUGCCAACAUAUUGCCAUC28 AGGCAAGAUGCUGGCAUAGC 15 UGUU AGGCAAGAUGCUGGCAU 16 AGGCAAGAUGCUGGCAUA17 AGGCAAGAUGCUGGCA 18 GGCAAGAUGCUGGCAUAGCU 19 G GGCAAGAUGCUGGCAUAGCU 20GGCAAGAUGCUGGCAUAGCU 21 GUU GGCAAGAUGCUGGCAUAGCU 22 GU As used herein,thymine (T) and uracil (U) may be used interchangeably depending on therespectivetype of polynucleotide.

Identity, moreover, means that there is preferably a functional and/orstructural equivalence between the corresponding nucleotide sequences.Nucleic acid sequences having the given identity levels to theparticular nucleic acid sequences of the polynucleotides to be inhibitedin context of the present invention may represent derivatives/variantsof these sequences which, preferably, have the same biological function.In context of the present invention, the biological function of apolynucleotide to be inhibited in context of the present invention isthe ability to decrease or suppress expression of FZD3 or a biologicallyactive derivative thereof, e.g., by hybridizing to the mRNA of FZD3,thereby inducing degradation or preventing translation of the FZD3 mRNA.Whether the expression of FZD3 or a biologically active derivativethereof has been decreased or suppressed can be easily tested by methodswell known in the art and as also described herein. Examples of suchmethods suitable to determine whether the expression of FZD3 or abiologically active derivative is decreased or suppressed arepolyacrylamide gel electrophoresis and related blotting techniques suchas Western Blot paired with chromogenic dye-based protein detectiontechniques (such as silver or coomassie blue staining) or withfluorescence- and luminescence-based detection methods for proteins insolutions and on gels, blots and microarrays, such as immunostaining, aswell as immunoprecipitation, ELISA, microarrays, and mass spectrometry.To determine whether a given polynucleotide hybridizes to the mRNA ofFZD3 can also be tested by methods well known in the art and as alsodescribed herein. Examples of such methods suitable to determine whethera given polynucleotide hybridizes to another nucleic acid (e.g., themRNA of FZD3 or a biologically active derivative thereof) are reportergene assays in which commonly used reporter genes are fluorescentproteins such as GFP, eGFP, YFP, eYFP, BFP, eBFP, luminescent proteinssuch as the enzymes Renilla or firefly luciferase, and β-galactosidaseencoded by the lacZ gene (Inui, Nat Rev Mol Cell Biol (2010), 11:252-63). Whether the mRNA of FZD3 is degraded or its translation isprevented can also be tested by methods known in the art and as alsodescribed herein. Examples for methods suitable to determine whether anmRNA is degraded are qPCR, RT-PCR, qRT-PCR, RT-qPCR, Light Cycler®,TaqMan® Platform and Assays or quantigene assay (Zhou, Anal Biochem(2000), 282: 46-53) Northern blot, dot blot, RNAse protection assays,microarrays, next generation sequencing (VanGuilder, Biotechniques(2008), 44(5): 619-26; Elvidge, Pharmacogenomics (2006), 7: 123-134;Metzker, Nat Rev Genet (2010), 11: 31-46; Kafatos, NAR (1979), 7:1541-1552).

The polynucleotides described herein, e.g. those to be inhibited incontext of the present invention may be either naturally occurringvariations, for instance sequences from other varieties, species, etc.,or mutations, and said mutations may have formed naturally or may havebeen produced by deliberate mutagenesis. Furthermore, the variations maybe synthetically produced sequences. The allelic variants may benaturally occurring variants or synthetically produced variants orvariants produced by recombinant DNA, RNA, PNA, GNA, TNA or LNAtechniques known in the art. Deviations from the above-described nucleicacid sequences may have been produced, e.g., by deletion, substitution,addition, insertion of nucleotides and/or by recombination. The term“addition” refers to adding at least one nucleic acid residue to one orboth ends of the given sequence, whereas “insertion” refers to insertingat least one nucleic acid residue within a given nucleotide sequence.The term “deletion” refers to deleting or removal of at least onenucleic acid residue in a given nucleotide sequence. The term“substitution” refers to the replacement of at least one nucleic acidresidue in a given nucleotide sequence. The definitions forpolynucleotides to be inhibited above and below apply mutatis mutandisfor all nucleic acid molecules and polynucleotides provided anddescribed herein including those acting as an antagonist/inhibitor.

The polynucleotides described herein, e.g., those to be inhibited incontext of the present invention (i.e. polynucleotides which decreasesor suppresses expression of FZD3) or those acting asantagonists/inhibitors, may be nucleic acid analogues such as DNAmolecules, RNA molecules, oligonucleotide thiophosphates, substitutedribo-oligonucleotides, LNA molecules, PNA molecules, GNA (glycol nucleicacid) molecules, TNA (threose nucleic acid) molecules, morpholinopolynucleotides, or antagomir (cholesterol-conjugated; forantagonists/inhibitors) polynucleotides. Furthermore, in context of thepresent invention, the term “polynucleotide” as well as the term“nucleic acid molecule” may refer to nucleic acid analogues such as DNAmolecules, RNA molecules, oligonucleotide thiophosphates, substitutedribo-oligonucleotides, LNA molecules, PNA molecules, GNA (glycol nucleicacid) molecules, TNA (threose nucleic acid) molecules, morpholinopolynucleotides, or antagomir (cholesterol-conjugated; forantagonists/inhibitors) polynucleotides or hybrids thereof or anymodification thereof as known in the art (see, e.g., U.S. Pat. No.5,525,711, U.S. Pat. No. 4,711,955, U.S. Pat. No. 5,792,608 or EP 302175for examples of modifications). Nucleic acid residues comprised by thepolynucleotides may be naturally occurring nucleic acid residues orartificially produced nucleic acid residues. Examples for nucleic acidresidues are adenine (A), guanine (G), cytosine (C), thymine (T), uracil(U), xanthine (X), and hypoxanthine (HX). In context of the presentinvention, thymine (T) and uracil (U) may be used interchangeablydepending on the respective type of polynucleotide. For example, as theskilled person is aware of, a thymine (T) as part of a DNA correspondsto an uracil (U) as part of the corresponding transcribed mRNA. Thepolynucleotides may be single- or double-stranded, linear or circular,natural or synthetic, and, if not indicated otherwise, without any sizelimitation. For instance, the polynucleotide to be inhibited in contextof the present invention may be a microRNA (miRNA) or a precursorthereof, a mimic microRNA or a precursor thereof, an siRNA or aprecursor thereof, a long non-coding RNA or a precursor thereof, ansnRNA (small/short hairpin RNA) or a precursor thereof, an stRNA (smalltemporal RNA) or a precursor thereof, an fRNA (functional RNA) or aprecursor thereof, an snRNA (small nuclear RNA) or a precursor thereof,a snoRNA (small nucleolar RNA) or a precursor thereof, a piRNA(piwi-interacting RNA) or a precursor thereof, a tasiRNA (trans-actingsmall/short interfering RNA) or a precursor thereof, an aRNA (antisenseRNA) or a precursor thereof, or a small non-coding RNA or a precursorthereof, genomic DNA, cDNA, mRNA, ribozymal or a DNA encoding the beforementioned RNAs or chimeroplasts (Gamper, Nucleic Acids Research (2000),28, 4332-4339). As already described, as used herein, “precursors” ofthe polynucleotides to be inhibited in context of the present inventionmay be forms of the respective polynucleotides as they occur duringmaturation of the respective polynucleotides. For example, in context ofthe present invention, precursors of a microRNA or a mimic microRNA maybe primary miRNAs (pri-miRNAs) or precursor miRNAs (pre-miRNAs) asoccurring during maturation of miRNAs. Both are single transcripts (i.e.ssRNA) that fold into a characteristic intramolecular secondarystructure, the so-called “hairpin loop”, which contains a stretch ofabout 18 to 23 base pairs, which is often interrupted by mismatches. Incontext of the present invention, precursors of siRNAs may be long dsRNAmolecules or shorter “hairpin loop” ssRNA molecules. Both types of thesesiRNA precursors may contain a stretch of base pairs without anymismatch. The current model for maturation of mammalian miRNAs isnuclear cleavage of the primary miRNA (pri-miRNA) which liberates a60-70 nt stem loop intermediate, known as the miRNA precursor orpre-miRNA. The mature about 18-23 nt long miRNA is yielded from one armof the stem loop precursor (Bartel, Cell (2004), 116: 281-297; Lee, EMBOJ (2002), 21: 4663-4670; Zeng and Cullen, RNA (2003), 9: 112-123). Saidpolynucleotides may be in the form of a plasmid or of viral DNA or RNA.Preferably, the polynucleotide to be inhibited in context of the presentinvention is a microRNA or a mimic microRNA.

In one embodiment, the polynucleotide to be inhibited in context of thepresent invention comprises or consists of the nucleotide sequence ofany one of SEQ ID NO: 1 (mature miR-31), SEQ ID NO: 2 (seed sequence ofmiR-31), or SEQ ID NO: 3 (pre-miR-31) as also shown in Table 1 herein.Furthermore, a polynucleotide to be inhibited in context of the presentinvention may also have a nucleic acid sequence comprising or consistingof a nucleotide sequence consisting of the seed sequence of SEQ ID NO: 1or SEQ ID NO: 3 as shown in Table 1 including one, two, three or morenucleotide(s) of the corresponding mature- or pre-miR sequence at the5′-end and/or the 3′-end of the respective seed sequence. For example, apolynucleotide to be inhibited in context of the present invention mayhave a nucleic acid sequence comprising or consisting of the nucleotidesequence [A] G-G-C-A-A-G-A-U [GC] (i.e. the seed sequence of SEQ ID NO:1 plus one nucleotide of the corresponding mature miRNA sequence at the5′-end and two nucleotides of the corresponding mature miRNA sequence atthe 3′-end) or [A] G-G-C-A-A-G-A-U [G] (i.e. the seed sequence of SEQ IDNO: 1 plus one nucleotide of the corresponding mature sequence at the5′-end and one nucleotide of the corresponding mature sequence at the3′-end). Polynucleotides to be inhibited in context of the presentinvention (i.e. polynucleotides which decrease or suppress expression ofFZD3) may also comprise or consist of the nucleotide sequence shown inany one of SEQ ID NO: 1 (mature miR-31), SEQ ID NO: 2 (seed sequence ofmiR-31), or SEQ ID NO: 3 (pre-miR-31) as also shown in Table 1 herein,wherein one, two, three, four, five or more nucleotides are added,deleted or substituted. Furthermore, a polynucleotide to be inhibited incontext of the present invention may also have a nucleic acid sequencecomprising or consisting of the nucleotide a nucleotide sequenceconsisting of the seed sequence of SEQ ID NO: 1 or SEQ ID NO: 3 as shownin Table 1 including one, two, three or more nucleotide(s) of thecorresponding mature- or pre-miR sequence at the 5′-end and/or the3′-end of the respective seed sequence, wherein one, two, three, four,five or more nucleotides are added, deleted or substituted. For example,a polynucleotide to be inhibited in context of the present invention mayhave a nucleic acid sequence comprising or consisting of the nucleotidesequence [A] G-G-C-A-A-G-A-U [U] (i.e. the seed sequence of SEQ ID NO: 1plus one nucleotide of the corresponding mature sequence at the 5′-endand one nucleotide of the corresponding mature sequence at the 3′-end,wherein the nucleotide at the 3′-end has been substituted by U).Preferably, said addition, deletion or substitution of one, two, three,four, five or more nucleotides is not effected within the seed sequenceof a polynucleotide as shown in Table 1 herein. Also, the polynucleotideto be inhibited in context of the present invention may comprise orconsist of a polynucleotide being at least 80%, 85%, 90%, 95%, 97%, 98%or 99% identical to a polynucleotide comprising or consisting of thenucleotide sequence of any one of SEQ ID NO: 1 (mature miR-31), SEQ IDNO: 2 (seed sequence of miR-31) or SEQ ID NO: 3 (pre-miR-31) as alsoshown in Table 1 herein. Furthermore, a polynucleotide to be inhibitedin context of the present invention may also comprise or consist of anucleic acid sequence with an identity of at least 80%, 85%, 90%, 95%,97%, 98% or 99% to a polynucleotide comprising or consisting of anucleotide sequence consisting of the seed sequence of SEQ ID NO: 1 orSEQ ID NO: 3 as shown in Table 1 including one, two or morenucleotide(s) of the corresponding mature- or pre-miR sequence at the5′-end and/or the 3′-end of the respective seed sequence. For example, apolynucleotide to be inhibited in context of the present invention maycomprise or consist of a nucleic acid sequence with an identity of atleast 80%, 85%, 90%, 95%, 97%, 98% or 99% to a polynucleotide comprisingor consisting of the nucleotide sequence G-G-C-A-A-G-A-U [GC] (i.e. theseed sequence of SEQ ID NO: 1 plus two nucleotides of the correspondingmature sequence at the 3′-end). Additionally, a polynucleotide to beinhibited in context of the present invention may also comprise orconsist of a nucleic acid sequence with an identity of at least 80%,85%, 90%, 95%, 97%, 98% or 99% to a polynucleotide comprising orconsisting of the nucleotide sequence of any one of SEQ ID NO: 1 (maturemiR-31) or SEQ ID NO: 3 (pre-miR-31) as also shown in Table 1 herein andcomprise the nucleic acid sequence as shown in SEQ ID NO: 2 (seedsequence of miR-31) as shown in Table 1 herein.

Generally, as used herein, a polynucleotide comprising the nucleic acidsequence of a sequence provided herein may also be a polynucleotideconsisting of said nucleic acid sequence. In one embodiment, thepolynucleotide to be inhibited in context of the present invention hasthe nucleic acid sequence as shown in SEQ ID NO: 1.

In context of the determination whether two given nucleic acid moleculesare able to hybridize, e.g., whether a polynucleotide to be inhibited incontext of the present invention hybridizes to an mRNA of FZD3 or abiologically active derivative thereof, or whether an inhibitordescribed in and used in accordance with the present inventionhybridizes to a polynucleotide to be inhibited in context of the presentinvention, the hybridization may occur and be detected underphysiological or artificial conditions, under stringent or non-stringentconditions. Said hybridization conditions may be established accordingto conventional protocols described, for example, in Sambrook, Russell“Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory,N.Y. (2001); Ausubel, “Current Protocols in Molecular Biology”, GreenPublishing Associates and Wiley Interscience, N.Y. (1989), or Higginsand Hames (Eds.) “Nucleic acid hybridization, a practical approach” IRLPress Oxford, Washington D.C., (1985). The setting of conditions is wellwithin the skill of the artisan and can be determined according toprotocols described in the art. Thus, the detection of only specificallyhybridizing sequences will usually require stringent hybridization andwashing conditions such as 0.1×SSC, 0.1% SDS at 65° C. Non-stringenthybridization conditions for the detection of homologous or not exactlycomplementary sequences may be set at 6×SSC, 1% SDS at 65° C. As is wellknown in the art, the length of the probe and the composition of thenucleic acid to be determined constitute further parameters of thehybridization conditions. Variations in the above conditions may beaccomplished through the inclusion and/or substitution of alternateblocking reagents used to suppress background in hybridizationexperiments. Typical blocking reagents include Denhardt's reagent,BLOTTO, heparin, denatured salmon sperm DNA, and commercially availableproprietary formulations. The inclusion of specific blocking reagentsmay require modification of the hybridization conditions describedabove, due to problems with compatibility. In accordance to theinvention described herein, low stringent hybridization conditions forthe detection of homologous or not exactly complementary sequences may,for example, be set at 6×SSC, 1% SDS at 65° C. As is well known in theart, the length of the probe and the composition of the nucleic acid tobe determined constitute further parameters of the hybridizationconditions. Polynucleotides to be inhibited in context of the presentinvention which hybridize to the mRNA of FZD3 or a biologically activederivative thereof also comprise fragments of the above describedpolynucleotides which are to be inhibited in context of the presentinvention. Such fragments preferably are polynucleotides which are ableto decrease or suppress expression of FZD3 or a biologically activederivative thereof. Such fragments may be, e.g., polynucleotides such assiRNAs or siRNA pools consisting of 4 siRNAs targeting the mRNA of FZD3as can be purchased from Dharmacon (on-target plus smart-poolL-005502-00-0005, NM_(—)017412). Furthermore, a hybridization complexrefers to a complex between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T (or U, respectively) bases; these hydrogenbonds may be further stabilized by base stacking interactions. Ahybridization complex may be formed in solution (e.g., Cot or Rotanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which, e.g., cellshave been fixed). The terms complementary or complementarity refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence “A-G-T(or U, respectively)” binds to the complementary sequence “T (or U,respectively)-C-A”. Complementarity between two single-strandedmolecules may be “partial”, in which only some of the bases of thenucleic acids bind, or it may be complete when total complementarityexists between single-stranded molecules. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands.

In order to determine whether two nucleic acid molecules hybridize,e.g., whether a given polynucleotide hybridizes to the mRNA of FZD3 or abiologically active derivative thereof as described herein, therebyinducing degradation or preventing translation of said mRNA of FZD3 or abiologically active derivative thereof, or whether an inhibitordescribed in and used in accordance with the present inventionhybridizes to a polynucleotide to be inhibited in context of the presentinvention, various tests known in the art and also described herein maybe applied. In this context, the hybridization may occur and be testedunder physiological conditions or under artificial conditions as knownin the art and also described herein. For example, a test to determinehybridization between an miRNA and an mRNA may be a Luciferase Assay asalso described herein and in technical bulletins by Promega (C8021(psiCHECK-2 Vector), E1960 (Dual-Luciferase® Reporter Assay System)). Incontext of the present invention, general examples of methods suitableto determine whether a polynucleotide hybridizes to another nucleic acid(e.g., the 3′UTR of the mRNA of FZD3) are reporter gene assays in whichcommon reporter genes are used such as fluorescent proteins (e.g., GFP,eGFP, YFP, eYFP, BFP, or eBFP), or luminescent proteins (e.g., Renillaor firefly luciferase, or 3-galactosidase encoded by the lacZ gene).Furthermore, degradation of mRNA or the level of the respectivetranslation product (to test whether the translation of the mRNA wasdecreased or prevented) can easily be examined by methods known in theart. Examples for methods suitable to examine degradation orstabilization of mRNA are qPCR, RT-PCR, qRT-PCR, RT-qPCR, Light Cycler®,TaqMan® Platform and Assays, Northern blot, dot blot, microarrays, nextgeneration sequencing (VanGuilder, Biotechniques (2008), 44: 619-26;Elvidge, Pharmacogenomics (2006), 7: 123-134; Metzker, Nat Rev Genet(2010), 11: 31-46). Examples for methods suitable to examine whether thetranslation of a mRNA has been prevented or decreased are polyacrylamidegel electrophoresis and related blotting techniques such as Western Blotpaired with chromogenic dye-based protein detection techniques (such assilver or coomassie blue staining) or with fluorescence- andluminescence-based detection methods for proteins in solutions and ongels, blots and microarrays, such as immunostaining, as well asimmunoprecipitation, ELISA, microarrays, and mass spectrometry (WesternBlot (Burnette, Anal Biochem (1981) 112: 195-203) or ELISA (Crowther,JA. The ELISA Guidebook. Humana Press; Totowa, N.J.: 2001).

In context of the present invention, in order to determine whether agiven polynucleotide decreases or suppresses expression of FZD3 or abiologically active derivative thereof (e.g., by hybridizing to the mRNAof FZD3 and thereby inducing degradation or preventing translation ofFZD3 mRNA), the level of expressed FZD3 can be easily detected. Incontext of the present invention, a polynucleotide is to be assessed asdecreasing or suppressing expression of FZD3 or a biologically activederivative thereof if the detected level of expressed FZD3 in a testsample which was contacted with a polynucleotide to be tested is atleast 1.5 fold, preferably at least L75 fold, more preferably at least2.0 fold, and most preferably at least 2.5 fold lower than the FZD3expression level of a control sample which was not contacted with thepolynucleotide. For example, a Western blot analysis can be performedfor FZD3 protein detection.

Furthermore, in one embodiment, the polynucleotides to be inhibited incontext of the present invention may hybridize to the 3′UTR(untranslated region) of the mRNA of FZD3 or a biologically activederivative thereof or to fragments of said 3′UTR. Hybridization betweena polynucleotide to be inhibited in context of the present invention andthe 3′UTR of the mRNA of FZD3 or a biologically active derivativethereof can easily be tested as described hereinabove. Preferably, byhybridizing to the 3′UTR of the mRNA of FZD3 or a biologically activederivative thereof or to fragments of said 3′UTR, the polynucleotide tobe inhibited in context of the present invention induces degradation ofor prevents translation of said mRNA of FZD3 or a biologicallyderivative thereof. Generally, in context of the present invention, whenreferring to FZD3, reference is made to GenBank Accession No.NM_(—)017412, Version No. 177 (released on Apr. 15, 2010). The sequenceof the 3′UTR of FZD3 mRNA is shown in SEQ ID NO: 4 herein. In oneembodiment, the polynucleotide to be inhibited in context of the presentinvention is able to hybridize to a nucleic acid sequence comprisingnucleotides 3419-3426 of SEQ ID NO: 4, preferably thereby inducingdegradation of or prevention of translation of the mRNA of FZD3 or abiologically derivative thereof.

The polynucleotide to be inhibited in context of the present inventionmay be comprised in lipid composition, an exosome, a vesicular body, aliposome, in PEI (polyethylene imine) or atellocollagen. Also, inaccordance with the present invention, the antagonist/inhibitor of apolynucleotide to be inhibited (i.e. that is capable of decreasing orsuppressing expression of FZD3 or a biologically derivative thereof) maybe comprised in a lipid composition, an exosome or a liposome. Forexample, an antagonist/inhibitor may be an antagonist/inhibitor ofmiR-31 or its 3′ or 5′ isoforms or variants as described herein.Examples for 3′ and 5′ isoforms of miR-31 are shown in Table 3. Thepresent invention also relates to such lipid compositions, exosomes andliposomes for use in the medical interventions described herein, e.g.,for use in treating or preventing bone disorders and/or cardiovasculardisorders such as osteoporosis, osteopenia, bone fracture, impaired bonehomeostasis, or cardiovascular diseases such as stroke, infarction,hypertension, thrombosis, vascular stenosis, coronary syndromes,vascular dementia, heart and renal failure or atherosclerosis in asubject.

As used herein, a biologically active derivative of FZD3 means that ishas the same biological function as FZD3, i.e. it is able to transducesignals via the PI3K-AKT pathway (Kawasaki, Cell Signal (2007), 19:2498-506). In context of the present invention, in order to validatewhether a given compound is a biologically derivative of FDZ3, thephosphorylation status of its downstream target AKT can be tested. Forthis purpose, ASCs, bone marrow derived stem cells, or any other celltype that has the ability to undergo osteogenic differentiation, may betreated with compounds to be tested for FZD3-activity for differenttimes and with different doses, or the cells may be transfected withplasmids coding for compounds to be tested for FZD3-activity. Then, celllysates may be prepared by methods known in the art. This can be done,for example, by using cell lysis buffer (20 mM Tris-HCl (pH 7.5)), 12 mM(3-glycerophosphate, 150 mM NaCl, 5 mM EGTA, 10 mM sodium fluoride, 1%Triton X-100, 1% sodium deoxycholate, 1 mM dithiothreitol (DTT), 1 mMsodium orthovanadate, protease inhibitor cocktail (Roche, Switzerland),phosphatase inhibitor cocktail 1 and 2 (Sigma, St. Louis, Mo.).Subsequently, an analysis of phosphorylated proteins may be performed bymethods known in the art, e.g., by immunoblot analyses. As anillustrative example for carrying out an immunoblot analysis, 30 μg oftotal proteins may be subjected to SDS-PAGE and blotted onpolyvinylidene difluoride membranes (e.g., PVDF). The membranes may thenbe probed with anti-phospho-Akt (Ser473) antibody (Cell Signalling,Danvers, 9271) (1:500), and anti-phospho-Akt (Thr308) antibody (CellSignalling, Danvers, 9272) (1:500). As positive control, overexpressionof FZD3 can be used. As negative controls, specific PI3K inhibitors likeWortmannin or LY294002 can be used that are known to prevent AKTphosphorylation. In context of the present invention, if AKT isphosphorylated to an extent of more than 40% or more than 50% comparedto untreated cell controls, the compound rested can be considered abiologically active derivative of FZD3. The nucleic acid sequence of the3′UTR of the mRNA of a FZD3 derivative in context of the presentinvention may be at least 80%, 85%, 90%, 95% or 98% identical to SEQ IDNO: 4.

As already mentioned, the present invention relates to a compositioncomprising an inhibitor of a polynucleotide or polynucleotides to beinhibited, i.e, which are capable of decreasing or suppressingexpression of FZD3 or a biologically derivative thereof for use intreating or preventing bone disorders and/or cardiovascular disorderssuch as osteoporosis, osteopenia, bone fracture, impaired bonehomeostasis, or cardiovascular diseases such as stroke, infarction,hypertension, thrombosis, vascular stenosis, coronary syndromes,vascular dementia, heart and renal failure or atherosclerosis in asubject. The composition may also comprise an exosome and/or a liposomewhich contain an antagonist/inhibitor of a polynucleotide that iscapable of decreasing or suppressing expression of FZD3 or abiologically derivative thereof and/or which contain anantagonist/inhibitor of miR-31 or its 3′ or 5′ isoforms or variants.

In accordance with the present invention, the subject to be treated orin which a bone disorder and/or cardiovascular disorder such asosteoporosis, osteopenia, bone fracture, impaired bone homeostasis, orcardiovascular diseases such as stroke, infarction, hypertension,thrombosis, vascular stenosis, coronary syndromes, vascular dementia,heart and renal failure or atherosclerosis is to be prevented may bemammalian. In a preferred embodiment of the present invention, thesubject is human.

Generally, the composition to be used in context of the presentinvention may comprise an antagonist/inhibitor or exosome/liposomecontaining said antagonist/inhibitor which inhibits one, two, three ormore of the polynucleotides to be inhibited in context of the presentinvention. Also, the composition may comprise two, three or moreantagonists/inhibitors or exosomes/liposomes, wherein each of theantagonist/inhibitors is capable of inhibiting one, two, three or moreof the polynucleotides to be inhibited in context of the presentinvention.

The composition described herein and to be employed in context with thepresent invention may contain the antagonist/inhibitor orexosome/liposome described herein in an amount of about 1 ng/kg bodyweight to about 100 mg/kg body weight of the subject which is to betreated or in which a bone disorder and/or cardiovascular disorder suchas osteoporosis, osteopenia, bone fracture, impaired bone homeostasis,or cardiovascular diseases such as stroke, infarction, hypertension,thrombosis, vascular stenosis, coronary syndromes, vascular dementia,heart and renal failure or atherosclerosis is to be prevented. In apreferred embodiment of the present invention, the composition comprisesthe inhibitor in an amount of about 1 μg/kg body weight to about 20mg/kg body weight, more preferably 1 mg/kg body weight to about 10 mg/kgbody weight.

The composition described herein and to be employed in context of thepresent invention may further comprise a pharmaceutically acceptablecarrier. Accordingly, the present invention also relates to apharmaceutical composition comprising an antagonist/inhibitor of apolynucleotide or polynucleotides to be inhibited in context of thepresent invention, an antagonist/inhibitor of miR-31 or its 3′ or 5′isoforms or variants, and/or an exosome or liposome containing saidantagonist/inhibitor and further comprising a pharmaceuticallyacceptable carrier, excipient and/or diluent. Generally, examples ofsuitable pharmaceutical carriers are well known in the art and includephosphate buffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose, i.e. about 1 ng/kg bodyweight to about 100 mg/kg body weight of the subject which is to betreated or in which bone disorders and/or cardiovascular disorders suchas osteoporosis, osteopenia, bone fracture, impaired bone homeostasis,or cardiovascular diseases such as stroke, infarction, hypertension,thrombosis, vascular stenosis, coronary syndromes, vascular dementia,heart and renal failure or atherosclerosis are to be prevented. In apreferred embodiment of the present invention, the compositioncomprising an inhibitor of a polynucleotide or polynucleotides to beinhibited in context of the present invention comprises the inhibitor inan amount of about 1 μg/kg body weight to about 20 mg/kg body weight,more preferably 1 mg/kg body weight to about 10 mg/kg body weight.Administration of the composition may be effected or administered bydifferent ways, e.g., enterally, orally (e.g., pill, tablet (buccal,sublingual, orally, disintegrating, capsule, thin film, liquid solutionor suspension, powder, solid crystals or liquid), rectally (e.g.,suppository, enema), via injection (e.g., intravenously, subcutaneously,intramuscularly, intraperitoneally, intradermally) via inhalation (e.g.,intrabronchially), topically, vaginally, epicutaneously, orintranasally. In context of the present invention, compositionscomprising exosomes or liposomes containing an antagonist/inhibitor of apolynucleotide capable of decreasing or suppressing expression of FZD3or a biologically derivative thereof and/or an antagonist/inhibitor ofmiR-31 or its 3′ or 5′ isoforms or variants may be applied locally orsystemically. When applied locally, e.g. directly at a defect site of abone, the composition comprising an exosome/liposome is particularly foruse in treating bone disorders such as osteoporosis, osteopenia, bonefracture or impaired bone homeostasis. Methods for applying suchcompositions directly to a bone are known in the art (Takeshita, MolTher (2010), 18: 181-187). When applied systemically (e.g.,parenterally, orally or other routes described herein), said compositionmay be used for treating or preventing bone disorders and/orcardiovascular disorders as described herein. It is understood that alsothe antagonists/inhibitors described herein may be administered locallyor systemically by other means, even directly. The dosage regimen willbe determined by the attending physician and clinical factors. As iswell known in the medical arts, dosages for any one patient depends uponmany factors, including the patient's size, body surface area, age, theparticular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. The compositions described herein may be administeredlocally or systemically. Systemic administration will preferably beparenterally, e.g., intravenously. The composition may also beadministered directly to the target site, e.g., by biolistic delivery toan internal or external target site or by catheter to a site in anartery. Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, polyethylene imine and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like. Furthermore, also doses below orabove of the exemplary ranges described hereinabove are envisioned,especially considering the aforementioned factors.

As already mentioned, the compositions described herein comprising anantagonist/inhibitor of a polynucleotide or polynucleotides to beinhibited in context of the present invention being capable ofdecreasing or suppressing expression of FZD3 or a biologicallyderivative thereof, an antagonist/inhibitor of miR-31 or its 3′ or 5′isoforms or variants, and/or an exosome/liposome containing saidantagonist/inhibitor may be used to treat or prevent bone disordersand/or cardiovascular disorders such as osteoporosis, osteopenia, bonefracture, impaired bone homeostasis, or cardiovascular diseases such asstroke, infarction, hypertension, thrombosis, vascular stenosis,coronary syndromes, vascular dementia, heart and renal failure oratherosclerosis in a subject.

In context of the present invention, an antagonist/inhibitor of apolynucleotide or polynucleotides to be inhibited in accordance with thepresent invention may be a nucleic acid molecule, a polypeptide or anyother compound capable of antagonizing/inhibiting the polynucleotides tobe inhibited in context of the present invention. For example, theantagonist/inhibitor to be employed in context of the present inventionis an antagonist/inhibitor of miR-31 or its 5′ or 3′ isoforms orvariants. An antagonist/inhibitor to be employed in context of thepresent invention may be a nucleic acid molecule capable of hybridizingto the polynucleotide to be inhibited. As described herein, nucleic acidmolecules comprise all kinds of nucleotide molecules such as DNAmolecules, RNA molecules, oligonucleotide thiophosphates, substitutedribo-oligonucleotides, LNA molecules (Prom, Gene (2006), 10(372),137-141), PNA molecules, GNA (glycol nucleic acid) molecules, TNA(threose nucleic acid) molecules, morpholino polynucleotides, orantagomir (cholesterol-conjugated) polynucleotides as well asmodifications thereof. The antagonist/inhibitor may hybridize to saidpolynucleotide to be inhibited under stringent or non-stringentconditions as described herein (for hybridization conditions for shortsequences, see below), preferably under stringent conditions. Methodsfor determining and evaluating hybridization between nucleic acidmolecules are well known in the art and are also described herein aboveand below. Preferably, in accordance with the present invention, byhybridizing to a polynucleotide to be inhibited in context of thepresent invention, the antagonist/inhibitor prevents said polynucleotideto be inhibited from decreasing or suppressing expression of FZD3 or abiologically derivative thereof, e.g., by hybridization of saidpolynucleotide with the mRNA (e.g., the 3′UTR thereof) of FZD3 or abiologically derivative thereof. Methods for determining and evaluatingthe capability of a polynucleotide to decrease or suppress theexpression of FZD3 or a biologically active derivative thereof as wellas methods for determining or evaluating whether the expression level ofFZD3 is decreased or suppressed are described herein. Accordingly, agiven compound can be assessed as an antagonist/inhibitor to be employedin context of the present invention if it is able to preventhybridization of a polynucleotide to be inhibited in context of thepresent invention with the mRNA (e.g., the 3′UTR thereof) of FZD3 or abiologically derivative thereof. Accordingly, in context of the presentinvention, an antagonist/inhibitor may be able to at least partiallyreverse the effect of a polynucleotide to be inhibited in context of thepresent invention on the expression of FZD3 or a biologically activederivative thereof. For example, the antagonist/inhibitor to be employedin context of the present invention may be capable of reversing theeffect of a polynucleotide to be inhibited on FZD3-expression by 50% ormore, preferably by 60% or more, more preferably by 70% or more, morepreferably by 80% or more, more preferably by 90% or more, morepreferably by 95% or more, more preferably by 98% or more, and mostpreferably by 99% or more. That is, e.g., if a polynucleotide to beinhibited in context of the present invention is capable of decreasingor suppressing the expression of FZD3 or a biologically derivativethereof such as it amounts to an expression level of 50% compared to thenormal expression level (i.e. without said polynucleotide), and theexpression level increases by applying an inhibitor as described hereinsuch that the expression level of FZD3 or a biologically derivativethereof increases to an amount of 75% compared to the normal expressionlevel (i.e. without said polynucleotide), the effect of saidpolynucleotide is reversed by said inhibitor by 50%.

In one embodiment of the present invention, the antagonist/inhibitor ofa polynucleotide to be inhibited in context of the present invention isa nucleic acid molecule which is capable of hybridizing to saidpolynucleotides to be inhibited, preferably under stringent conditionsas described herein, thereby preventing said polynucleotide fromhybridizing to the mRNA (e.g., the 3′UTR thereof) of FZD3 or abiologically active derivative thereof. The hybridization of saidnucleic acid to be employed as an antagonist/inhibitor to apolynucleotide to be inhibited in context of the present invention maybe over the entire length of said polynucleotide to be inhibited or onlyover a part of the sequence of said polynucleotide to be inhibited,e.g., over at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% of the sequence ofsaid polynucleotide to be inhibited. In one embodiment of the presentinvention, the antagonist/inhibitor to be employed in context of thepresent invention may be an antisense oligonucleotide which iscomplementary to a polynucleotide to be inhibited in context of thepresent invention. Preferably, in accordance with the present invention,an antagonist/inhibitor to be employed in context of the presentinvention is an antisense oligonucleotide which comprises or consists ofa nucleic acid molecule having a sequence complementary to any one ofthe sequences as shown in Table 1 hereinabove, e.g., to any one of SEQID NOs. 1 to 3. For example, such antisense oligonucleotides maycomprise or consist of a nucleotide sequence of any one of SEQ ID NOs. 5to 8. Generally, antagonists/inhibitors of miRNAs or siRNAs are wellknown in the art and customized miRNA- or siRNA-inhibitors arecommercially available. For example, antagonists/inhibitors ofpolynucleotides to be inhibited in context of the present invention maybe nucleic acid molecules such as antagomiRs (Krüzfeldt, Nature (2005),438: 685-689) or any other 2′-O-methyl-RNA oligonucleotide havingphosphorothioates bonds and a cholesterol tail, miRCURY LNA™ microRNAinhibitors (Exiqon), in vivo LNA™ miR inhibitors (Exiqon), tiny LNAs(Obad, Nat Genet (2011), 43(4): 371-378), miR-decoys or miR-sponges(Ebert, Nat Methods (2007), 4: 721-726; Bonci, Nat Med (2008), 14:1271-1277) or the like which are capable of antagonizing/inhibiting apolynucleotide to be inhibited in context of the present invention asdescribed hereinabove, e.g., by hybridizing to said polynucleotide. Anantagonist/inhibitor might also be or derive from miRNA degradingenzymes as described in Chatterjee, Nature (2009), 461: 546-9,hammerhead ribozymes as described in Tedeschi, Drug Discov Today (2009),14: 776-783, or antogomirzymes as described in Jadhav, Angew Chem Int EdEngl (2009), 48(14): 2557-2560. An example of an miRCURY LNA™ microRNAinhibitor in context of the present invention is anti-miR-31 LNA asshown in SEQ ID NO: 5. In context of the present invention, theantagmoiRs, miCURY LNA™ microRNA inhibitors, in vivo LNA™ miRinhibitors, tiny LNAs, miR decoys or miR sponges may comprise a nucleicacid molecule or include a nucleic acid sequence selected from the groupconsisting of

-   (a) a nucleic acid sequence being or comprising SEQ ID NO: 5;-   (b) a nucleic acid sequence being or comprising SEQ ID NO: 6;-   (c) a nucleic acid sequence being or comprising SEQ ID NO: 7;-   (d) a nucleic acid sequence being or comprising SEQ ID NO: 8; and-   (e) a nucleic acid sequence which is at least 90% or at least 95%    identical to the nucleic acid sequence of any one of (a) to (d).

As mentioned, such antagonists/inhibitors may hybridize or bind to allkinds of polynucleotides to be inhibited as described herein, includingmicroRNA, siRNA, mimic microRNA, long non-coding RNAs, snRNA, stRNA,fRNA, snRNA, snoRNA, piRNA, tasiRNA, aRNA and precursors of suchpolynucleotides.

The present invention relates to a composition comprising a nucleic acidmolecule which hybridizes under stringent conditions with thepolynucleotide consisting of the sequence shown in SEQ ID NO: 1, therebypreventing hybridization of said polynucleotide with the mRNA of FZD3,for use in treating or preventing bone disorder, like osteoporosis orcardiovascular disease, like atherosclerosis in a human subject.

The present invention relates to a composition comprising a nucleic acidmolecule which hybridizes under stringent conditions to miR-31 or its 3′or 5′ isoforms or variants, thereby preventing hybridization of miR-31or its 3′ or 5′ isoforms or variants with the mRNA of FZD3, for use intreating or preventing osteoporosis or atherosclerosis in a humansubject. Said nucleic acid molecule may be an antagomiR, a miCURY LNA™microRNA inhibitor, an in vivo LNA™ miR inhibitor, a miR decoy or a miRsponge as described herein.

The present invention relates to a composition comprising a nucleic acidmolecule which hybridizes under stringent conditions with thepolynucleotide consisting of the sequence shown in SEQ ID NO: 2, therebypreventing hybridization of said polynucleotide with the mRNA of FZD3,for use in treating or preventing osteoporosis or cardiovasculardiseases such as atherosclerosis in a human subject.

The present invention relates to a composition comprising a nucleic acidmolecule which hybridizes under stringent conditions with thepolynucleotide consisting of the sequence shown in SEQ ID NO: 3, therebypreventing hybridization of said polynucleotide with the mRNA of FZD3,for use in treating or preventing osteoporosis or cardiovasculardiseases such as atherosclerosis in a human subject.

In context of the composition of the preceding paragraphs, a nucleicacid molecule which hybridizes under stringent conditions with thepolynucleotide consisting of the nucleic acid sequence shown in any oneof SEQ ID NOs: 1 to 3 is an antagomiR.

In context of the composition of the preceding paragraphs, a nucleicacid molecule which hybridizes under stringent conditions with thepolynucleotide consisting of the nucleic acid sequence shown in any oneof SEQ ID NOs: 1 to 3 is a 2′-O-methyl-RNA oligonucleotide havingphosphorothioates bonds and a cholesterol tail.

In context of the composition of the preceding paragraphs, a nucleicacid molecule which hybridizes under stringent conditions with thepolynucleotide consisting of the nucleic acid sequence shown in any oneof SEQ ID NOs: 1 to 3 is a miRCURY LNA™ microRNA inhibitor (Exiqon).

In context of the composition of the preceding paragraphs, a nucleicacid molecule which hybridizes under stringent conditions with thepolynucleotide consisting of the nucleic acid sequence shown in any oneof SEQ ID NOs: 1 to 3 is an in vivo LNA™ miR inhibitors (Exiqon).

In context of the composition of the preceding paragraphs, a nucleicacid molecule which hybridizes under stringent conditions with thepolynucleotide consisting of the nucleic acid sequence shown in any oneof SEQ ID NOs: 1 to 3 is a miR-decoy or sponge.

Furthermore, in accordance with the present invention, theantagonist/inhibitor (i.e. in case of a nucleic acidantagonist/inhibitor) of the polynucleotide to be inhibited in contextof the present invention may be cloned into a vector. The term “vector”as used herein particularly refers to plasmids, cosmids, viruses,bacteriophages and other vectors commonly used in genetic engineering.In a preferred embodiment, these vectors are suitable for thetransformation of cells, like fungal cells, cells of microorganisms suchas yeast or prokaryotic cells. In a particularly preferred embodiment,such vectors are suitable for stable transformation of bacterial cells,for example to transcribe the polynucleotide of the present invention.

Accordingly, in one aspect of the invention, the vector as provided isan expression vector. Generally, expression vectors have been widelydescribed in the literature. As a rule, they may not only contain aselection marker gene and a replication-origin ensuring replication inthe host selected, but also a promoter, and in most cases a terminationsignal for transcription. Between the promoter and the terminationsignal there is preferably at least one restriction site or a polylinkerwhich enables the insertion of a nucleic acid sequence/molecule desiredto be expressed.

It is to be understood that when the vector provided herein is generatedby taking advantage of an expression vector known in the prior art thatalready comprises a promoter suitable to be employed in context of thisinvention, for example expression of an inhibitor (i.e. in case of anucleic acid inhibitor) of a polynucleotide as described hereinabove,the nucleic acid construct is inserted into that vector in a manner theresulting vector comprises only one promoter suitable to be employed incontext of this invention. The skilled person knows how such insertioncan be put into practice. For example, the promoter can be excisedeither from the nucleic acid construct or from the expression vectorprior to ligation.

As a non-limiting example, the vector into which an antagonist/inhibitor(i.e. in case of a nucleic acid antagonist/inhibitor) of apolynucleotide to be inhibited in context of the present invention (i.e.which decreases or suppresses FZD3-expression) is cloned is anadenoviral, adeno-associated viral (AAV), retroviral, or nonviralminicircle-vector. Further examples of vectors suitable to comprise theinhibitor (i.e. in case of a nucleic acid inhibitor) of a polynucleotideto be inhibited in context of the present invention to form the vectordescribed herein are known in the art. For example, a vector into whichan inhibitor (i.e. in case of a nucleic acid inhibitor) of apolynucleotide to be inhibited in context of the present invention hasbeen cloned may be miR-Vec, a retroviral expression vector (Voorhoeve,Cell (2006), 124: 1169-1181).

In an additional embodiment, the inhibitor (in case of a nucleic acidinhibitor or the coding nucleic acid sequence of a peptide inhibitor) ofa polynucleotide to inhibited in context of the present invention and/orthe vector into which the polynucleotide described herein is cloned maybe transduced, transformed or transfected or otherwise introduced into ahost cell. For example, the host cell is a eukaryotic or a prokaryoticcell, for example, a bacterial cell. As a non-limiting example, the hostcell is preferably a mammalian cell. The host cell described herein isintended to be particularly useful for generating the inhibitor of apolynucleotide to be inhibited in context of the present invention.

Generally, the host cell described hereinabove may be a prokaryotic oreukaryotic cell, comprising an inhibitor of the polynucleotide to beinhibited in context of the present invention or the vector describedherein or a cell derived from such a cell and containing the nucleicacid construct or the vector described herein. In a preferredembodiment, the host cell comprises, i.e. is genetically modified withnucleic acid sequence of the inhibitor of the polynucleotide to beinhibited in context of the present invention or the vector describedherein in such a way that it contains the nucleic acid sequence of theinhibitor of a polynucleotide to be inhibited in context of the presentinvention integrated into the genome. For example, such host celldescribed herein may be a bacterial, yeast, or fungus cell. In oneparticular aspect, the host cell is capable to transcribe the nucleicacid sequence of an inhibitor of a polynucleotide which decreases orsuppresses expression of FZD3 or a biologically active derivativethereof in context of the present invention. An overview of examples ofdifferent corresponding expression systems to be used for generating thehost cell described herein is for instance contained in Methods inEnzymology 153 (1987), 385-516, in Bitter (Methods in Enzymology 153(1987), 516-544), in Sawers (Applied Microbiology and Biotechnology 46(1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996),500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), and inGriffiths, (Methods in Molecular Biology 75 (1997), 427-440). Thetransformation or genetically engineering of the host cell with apolynucleotide to be inhibited in context of the present invention orvector described herein can be carried out by standard methods, as forinstance described in Sambrook and Russell (2001), Molecular Cloning: ALaboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA; Methods inYeast Genetics, A Laboratory Course Manual, Cold Spring HarborLaboratory Press, 1990.

Furthermore, as already mentioned and in context of the presentinvention, it was surprisingly found that miR-31 is a valuable tool as abiomarker for aging and age-associated diseases such as osteoporosis,osteopenia, bone fracture, impaired bone homeostasis or cardiovasculardiseases such as stroke, infarction, hypertension, thrombosis, vascularstenosis, coronary syndromes, vascular dementia, heart and renal failureor atherosclerosis. That is, the polynucleotides to be inhibited incontext of the present invention may also serve as diagnostic orprognostic markers themselves and detection of said polynucleotides,e.g., by using compounds binding thereto, will be useful in diagnosingor predicting the progression of diseases or disorders such asosteoporosis, osteopenia, bone fracture, impaired bone homeostasis orcardiovascular diseases such as stroke, infarction, hypertension,thrombosis, vascular stenosis, coronary syndromes, vascular dementia,heart and renal failure or atherosclerosis in a subject. Accordingly,the present invention relates to a compound binding to a polynucleotideto be inhibited in context of the present invention, i.e. to apolynucleotide which is capable of decreasing or suppressing expressionof FZD3 or a biologically derivative thereof as described herein, foruse in diagnosing or predicting the progression of bone disorders and/orcardiovascular disorders such as osteoporosis, osteopenia, bonefracture, impaired bone homeostasis, or cardiovascular diseases such asstroke, infarction, hypertension, thrombosis, vascular stenosis,coronary syndromes, vascular dementia, heart and renal failure oratherosclerosis in a subject.

Hence, the present invention further relates to a composition comprising

-   -   (a) a polynucleotide capable of decreasing or suppressing        expression of FZD3 or a biologically active derivative thereof        as described hereinabove, and/or    -   (b) a nucleic acid molecule which hybridizes to a polynucleotide        capable of decreasing or suppressing expression of FZD3 or a        biologically active derivative thereof as described hereinabove,        and/or    -   (c) an agent that binds to a polynucleotide capable of        decreasing or suppressing expression of FZD3 or a biologically        active derivative thereof as described hereinabove,    -   for use in diagnosing bone disorders and/or cardiovascular        disorders such as osteoporosis, osteopenia, bone fracture,        impaired bone homeostasis, or cardiovascular diseases such as        stroke, infarction, hypertension, thrombosis, vascular stenosis,        coronary syndromes, vascular dementia, heart and renal failure        or atherosclerosis in a subject.

The present invention also relates to a composition comprising

-   -   (a) an agent capable of specifically interacting with (e.g.,        binding to) a polynucleotide being capable of decreasing or        suppressing expression of FZD3 or a biologically active        derivative thereof, and/or    -   (b) a nucleic acid molecule which hybridizes, preferably under        stringent conditions, to a polynucleotide being capable of        decreasing or suppressing expression of FZD3 or a biologically        active derivative thereof,    -   for use in diagnosing bone disorders and/or cardiovascular        disorders in a subject or for use in monitoring in vitro the        treatment success of a bone disorder and/or a cardiovascular        disorder as described herein.

The term “agent” as used herein, particularly in context with “agentinteracting with a polynucleotide being capable of decreasing orsuppressing expression of FZD3 or a biologically active derivativethereof” or “agent that binds to a polynucleotide capable of decreasingor suppressing expression of FZD3 or a biologically active derivativethereof” comprises specific proteins or protein fragments. Such proteinsor protein fragments may be, e.g., antibodies or fragments thereof,small molecule inhibitors or transcription factors or modifiedtranscription factors. Examples of such (modified) transcription factorsare (modified) zinc finger proteins. Methods for generating smallmolecule inhibitors of polynucleotides are known in the art (Davis,Antivir Chem Chemother (2011), 21(3): 117-128). The terms “antibody” and“antibody fragment” are used herein in the broadest sense and includes,but is not limited to, monoclonal and polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), chimericantibodies, CDR grafted antibodies, humanized antibodies, camelizedantibodies, single chain antibodies and antibody fragments and fragmentconstructs, e.g., F(ab′)₂ fragments, Fab-fragments, Fv-fragments, singlechain Fv-fragments (scFvs), bispecific scFvs, diabodies, single domainantibodies (dAbs) and minibodies which are capable of specificallyinteracting with or binding to polynucleotides to be inhibited asdescribed herein. Methods for producing antibodies againstpolynucleotides are well known in the art (see, e.g., Ye, Proc Nat AcadSci USA (2008), 105: 82-87).

Furthermore, the present invention relates to pharmaceuticalcompositions comprising a compound, e.g., a nucleic acid molecule or anantibody, binding to a polynucleotide to be inhibited in context of thepresent invention for use in diagnosing bone disorders and/orcardiovascular disorders such as osteoporosis, osteopenia, bonefracture, impaired bone homeostasis, or cardiovascular diseases such asstroke, infarction, hypertension, thrombosis, vascular stenosis,coronary syndromes, vascular dementia, heart and renal failure oratherosclerosis in a subject. In one embodiment of the presentinvention, the compound binding to a polynucleotide to be inhibited incontext of the present invention is a nucleic acid molecule as describedin context of an inhibitor capable of hybridizing to said polynucleotideas described hereinabove. Hybridization of such a binding nucleic acidmolecule with said polynucleotide to be inhibited in context of thepresent invention can be easily detected by the skilled person usingmethods well known in the art and as also described herein. In oneembodiment, said binding compound is a binding agent such as an antibodyor a fragment thereof (such as F(ab) or F(ab)₂ fragments) specificallybinding to said polynucleotide to be inhibited in context of the presentinvention, i.e. which is capable of decreasing or suppressing expressionof FZD3 or a biologically derivative thereof. Binding of an antibody ora fragment thereof to a polynucleotide can be easily detected by theskilled person using methods well known in the art such as ELISA, EIA orsimilar methods.

Described herein is a method for diagnosing bone disorders and/orcardiovascular disorders such as osteoporosis, osteopenia, bonefracture, impaired bone homeostasis, or cardiovascular diseases such asstroke, infarction, hypertension, thrombosis, vascular stenosis,coronary syndromes, vascular dementia, heart and renal failure oratherosclerosis in a subject, said method comprising the steps of:

-   -   (a) obtaining a biological sample from said subject which        comprises a polynucleotide capable of decreasing or suppressing        expression of FZD3 or a biologically active derivative thereof        as described hereinabove;    -   (b) contacting said sample with a nucleic acid molecule which        hybridizes to the polynucleotide of (a), or with an agent that        binds to a polynucleotide of (a);    -   (c) detecting and evaluating hybridization or binding signal of        the nucleic acid molecule of (b) or the agent of (b) with the        polynucleotide of (a); and    -   (d) comparing the detected and evaluated hybridization or        binding signal of (c) with that correspondingly detected and        evaluated hybridization or binding signal in a control sample,        wherein a stronger hybridization or a stronger binding signal in        the sample of the subject compared to that of said control        sample is indicative for a risk of developing or having a bone        disorder and/or cardiovascular disorder.

The present invention relates to a method for diagnosing bone disordersand/or cardiovascular disorders such as osteoporosis, osteopenia, bonefracture, impaired bone homeostasis, or cardiovascular diseases such asstroke, infarction, hypertension, thrombosis, vascular stenosis,coronary syndromes, vascular dementia, heart and renal failure oratherosclerosis in a subject, said method comprising the steps of:

-   -   (a) (i) contacting a biological sample from said subject with a        nucleic acid molecule which hybridizes (preferably under        stringent conditions) to a polynucleotide being capable of        decreasing or suppressing expression of FZD3 and/or which        hybridizes (preferably under stringent conditions) to miR-31 or        its 5′ or 3′ isoforms or variants, or        -   (ii) contacting said biological sample with an agent that            binds to said polynucleotide being capable of decreasing or            suppressing expression of FZD3 and/or that binds to miR-31            or its 5′ or 3′ isoforms or variants;    -   (b) detecting and evaluating the hybridization signal of the        nucleic acid molecule of (a)(i) or detecting and evaluating the        binding signal of the agent of (a)(ii) with said polynucleotide        and/or said miR-31 or its 5′ or 3′ isoforms or variants; and    -   (c) comparing the detected and evaluated hybridization signal of        (b)(a)(i) or comparing the detected and evaluated the binding        signal of (b)(a)(ii) with a correspondingly detected and        evaluated hybridization or binding signal in a control sample,        wherein a stronger hybridization or a stronger binding signal in        the sample of the subject compared to that of said control        sample is indicative for a risk of developing or having a bone        disorder and/or cardiovascular disorder.

In one embodiment, the method for diagnosing bone disorders and/orcardiovascular disorders such as osteoporosis, osteopenia, bonefracture, impaired bone homeostasis, or cardiovascular diseases such asstroke, infarction, hypertension, thrombosis, vascular stenosis,coronary syndromes, vascular dementia, heart and renal failure oratherosclerosis in a subject, said method comprising the steps of

-   -   (a) contacting a biological sample from said subject with a        nucleic acid molecule which hybridizes (preferably under        stringent conditions) to a polynucleotide being capable of        decreasing or suppressing expression of FZD3 and/or which        hybridizes (preferably under stringent conditions) to miR-31 or        its 5′ or 3′ isoforms or variants;    -   (b) detecting and evaluating the hybridization signal of the        nucleic acid molecule of (a) with said polynucleotide and/or        said miR-31 or its 5′ or 3′ isoforms or variants; and    -   (c) comparing the detected and evaluated hybridization signal        of (b) with a correspondingly detected and evaluated        hybridization signal in a control sample,        wherein a stronger hybridization signal in the sample of the        subject compared to that of said control sample is indicative        for a risk of developing or having a bone disorder and/or        cardiovascular disorder.

The nucleic acid molecules to be employed in the diagnosing methods ofthe present invention (i.e. hybridizing to a polynucleotide beingcapable of decreasing or suppressing expression of FZD3) may be of anykind of nucleic acid molecules as described herein. For example, thesenucleic acid molecules may be primers (e.g., for PCR techniques) orprobes (e.g., for microarray or blot assays such as dot blot, southernblot or northern blot). Primers can be used particularly in PCRtechniques as described and exemplified herein. Preferably, as known inthe art, one primer is complementary to a sequence of the 5′-end of thepolynucleotide to be detected (“forward primer”), while the other primeris complementary to a sequence of the 3′-end of the polynucleotide to bedetected (“reverse primer”). Primers for PCR techniques and the likeshould usually have a length of about 12 to 30 nucleotides, but they mayalso comprise more or less nucleotides if appropriate. The primers maynot be 100% complementary to the respective sequences of thepolynucleotide to be detected as long as it is still capable ofhybridising to the polynucleotide, preferably under stringent conditionsor such conditions as may be appropriate for the given primer asdescribed herein below. Furthermore, primers may be conjugated to markermolecules or tagging molecules as described herein such as fluorescentdyes excited and emitting at UV/VIS or infrared wavelengths like FITC,TRITC, Texas Red, Cy-dyes, alexa dyes (Bioprobes), or the like. Probescan be generated as known in the art and usually comprise a nucleotidesequence which is complementary to a sequence of the polynucleotide tobe detected. Probes are of particular useful for assays such asmicroarrays or blot assays (e.g., dot blot, southern blot, northernblot) as described and exemplified herein. The sequence of the probedoes not have to be 100% complementary to the respective sequence of thepolynucleotide to be detected as long as it is still capable ofhybridising to the polynucleotide, preferably under stringentconditions. Furthermore, probes may be conjugated to marker molecules ortagging molecules as described herein such as fluorescent dyes excitedand emitting at UV/VIS or infrared wavelengths like FITC, TRITC, TexasRed, Cy-dyes, alexa dyes (Bioprobes), or the like.

Hybridization conditions in context with the diagnosing methods providedherein, particularly for PCR techniques as described above, may be asfollows. SSC is 3 M NaCl and 300 mM Na₃Citrate, adjusted to pH 7.0 withHCl. For stringent conditions, 0.1 or 0.2×SSC at 65° C. is used,preferably with addition of SDS at 1%. For intermediate conditions, 1×or 2×SSC is used, and for non-stringent conditions, 4× or 6×SSC is used,preferably with 1% SDS. The temperature is generally 65° C. or lessdepending on the melting temperature of the probe-target complex. Asmentioned, in context with the diagnosis methods provided herein, thenucleic acid molecule may hybridize to the polynucleotide to be detected(i.e. polynucleotide being capable of decreasing or suppressingexpression of FZD3) under stringent conditions.

In accordance with the present invention, determining the meltingtemperature (T_(m)) of a nucleic acid duplex is generally useful formany applications such as PCR, binding assays, hybridization, and thelike. The T_(m) may be defined as the temperature where 50% of a givennucleotide (such as a microRNA) is in duplex with its reversecomplementary (i.e. exactly matching for formation of a double helix)sequence. T_(m) is influenced by a number of factors well known to theperson of ordinary skill, such as the length of the polynucleotide, thebase composition, the sequence, the possibility to form secondarystructures (such as the hairpin structures typical for micro RNAprecursor molecules), and environmental conditions, such as salt(generally monovalent cation) concentration, divalent cation (Mg²⁺)concentration, pH, and the presence of denaturing substances such ase.g. formamide.

In order to conduct hybridization in a PCR or any other test thatrequires binding of a nucleotide to its target form a double helix, theT_(m) of the nucleotide may be determined. A number of methods fordetermining T_(m) are known to the person of ordinary skill in the art.For instance, the empirical determination could be carried out byincluding in a buffer solution the nucleotide to be tested and its exactreverse complement, and raising the temperature from a low temperaturewhere annealing is perfect (such as room temperature or less) to a hightemperature (such as 95° C. or more) where annealing cannot take placeanymore and most of the nucleotide will be single and not annealed. Thechange from the single to the duplex form of the nucleotide may bemonitored by UV spectrometry. For example, the nucleotide and its exactfitting reverse complement may be dissolved in a buffer such as 0.1×SSC(15 mM NaCl, 1.5 mM Na₃Citrate, pH 7.0). UV absorbance (Abs) may bemeasured at 260 nm and the temperature (t) gradually changed from low tohigh to observe denaturation and optionally, vice versa to observereannealing. When plotting the resulting temperature versus absorbance,an S-shaped curve may be observed, having two absorbance plateaus. TheT_(m) may then be determined as the temperature exactly halfway betweenthe upper plateau and the lower plateau, or, alternatively, as themaximum of the first derivative dAbs/dt. (see, e.g., Thermal Analysis ofDNA by UV/Visible Spectrometry, Moore, GBC application note, GBCScientific Equipment Pty, Ltd, Braeside, Australia,http://www.gbcscientific.com/appnotes/uv_app_note_(—)002.pdf), see also“Oligo melting temperature” by Sigma-Aldrich, St. Lois, Mo., USA(http://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/learning-center/oligos-melting-temphtml).

Of course, as is well known to the person of skill in the art, the T_(m)can be experimentally determined by the same method for non-perfectlymatching nucleotides, for RNA/DNA duplexes, for nucleotidesincorporating modified or non-canonical bases (such as Inosine,generally used as a nucleotide intended to bind almost equally well anyof the four canonical nucleotide bases A, C, G, T or U), or nucleotidemolecules with altered backbones (e.g., locked nucleic acid—LNA, orphosphorodiamidate morpholino nucleotides; see, e.g., Summerton,Antisense Nucleic Acid Drug Dev (1997), 7(3): 187-195).

The T_(m) determined experimentally is only valid for the conditions atwhich it was measured. For other conditions (e.g., salt (monovalentcation) concentration, divalent cation concentration), formulas may beused that allow an estimation of the T_(m) under different conditions(see below).

Alternatively, the approximate T_(m) of a given nucleotide sequence canbe calculated. A number of methods are known to the person of ordinaryskill in the arts, such as the GC content method, the nearest neighbourmethod and others. Generally, the nearest neighbour method results inmore accurate estimations, although none of these methods is able topredict the T_(m) of a given nucleotide sequence with absolute accuracy,so that a few ° C. deviation (generally assumed to be 5-10° C.) must betaken into account. Accordingly, in most hybridization, annealing,primer extension or other methods that rely on the formation of anucleotide duplex, the temperature at which the experiment is performedis chosen about 5-10° C. below the theoretically calculated T_(m), inorder to ensure perfect duplex formation. Of course, as is well known inthe art of molecular biology, hybridization conditions may be optimized,e.g., by testing the hybridization/primer annealing/PCR or otherexperiment that relies on nucleotide duplex formation at varioustemperature and other conditions and determining the conditions at whichthe best result is obtained.

A simple way to calculate estimated T_(m) values is based on the GCcontent of the nucleotide sequence (see, e.g., for short nucleotidesequences, the “Wallace rule”, Wallace, Nucleic Acids Res (1979), 6:3543; for longer nucleotides, see below). Briefly, different equationsare used for calculating the estimated T_(m) of duplexes containing DNAand RNA, as RNA bases tend to pair more strongly, resulting in higherT_(m) values for the same sequence compared to DNA-DNA duplexes(generally, RNA-RNA duplexes are most stable, RNA-DNA hybrids are ofintermediate stability, and DNA-DNA duplexes are least stable for thesame nucleotide sequence). One simple equation is the “Wallace rule”,where the melting temperature is determined as two ° C. for each A/Tpair and 4° C. for each G/C pair in the sequence (Td=2(#A/T)+4(#C/G).This equation would results in an estimate for the melting temperatureat a situation where one nucleotide is membrane-bound at a saltconcentration of 0.9 M. Where both nucleotides forming the duplex are insolution the T_(m) would be slightly lower than calculated by thisformula (about 7-8° C. lower). For nucleotides longer than about 14 to20 bases, the methods described in Howley, J Biol Chem (1979), 254: 4876may be used. Briefly, for DNA-DNA duplexes, T_(m) is calculated asT_(m)=81.5+16.6 log [Na⁺]+41(GC %)−500/L−0.62 F where Na⁺ is the molarconcentration of monovalent cations, GC % is the fraction of GCnucleotides versus the total number of nucleotides (being a valuebetween 0 and 1), L is the nucleotide length, and F is the percentage offormamide if present in the solution (being a value between 0 and 100).When calculating estimated values for RNA-DNA hybrids or RNA-RNAduplexes, a slightly different formula is used (T_(m)=79.8+18.5 log[Na⁺]+58.4 (% GC)+11.8 (% GC)²−820/L−0.35 F), while for DNA-RNA hybrids(the RNA being the molecule in solution), the formula is T_(m)=79.8+18.5log [Na⁺]+58.4 (% GC)+11.8 (% GC)²−820/L−0.50 F.

An improved method for estimation of a T_(m) value is the nearestneighbour method, so called because it takes into account not only thepercentage of G/C interactions in the duplex, but also the nearestneighbour of each nucleotide, effectively adding up the binding energiesof each dinucleotide in the sequence. This method results in differentestimation of T_(m) for different nucleotide sequences having the same Gcontent, yielding a more precise estimation for T_(m) than theabove-mentioned GC content method (see for overview, e.g., SantaLucia,Proc Natl Acad Sci USA (1998), 95: 1460-1465; for DNA see, e.g.,Breslauer, Proc Natl Acad Sci USA (1986), 83, 3746-3750; for RNA see,e.g., Freier, Proc Natl Acad Sci (1986), 83, 9373-9377). By way ofexample, the formula T_(m)=(1000ΔH/A+ΔS+R*ln(C/4))−273.15+16.6 log [Na⁺]may be used, where ΔH (Kcal/mol) is the sum of the nearest-neighbourenthalpy changes for hybrids, A is a small constant containingcorrections for helix initiation, ΔS is the sum of the nearest-neighbourentropy changes, R is the Gas Constant (1.99 cal/(K*mol)), C is theconcentration of the nucleotide, and Na⁺ is the concentration ofmonovalent cations in the solution. The ΔH and ΔS values to be used thiscalculation for DNA and RNA duplexes are shown in the table (formulaexample and values from Sigma-.Aldrich, reference see above).

Thermodynamic parameters for nearest-neighbour melting temperatureformula.

DNA RNA Interaction ΔH ΔS ΔH ΔS AA/TT −9.1 −24.0 −6.6 −18.4 AT/TA −8.6−23.9 −5.7 −15.5 TA/AT −6.0 −16.9 −8.1 −22.6 CA/GT −5.8 −12.9 −10.5−27.8 GT/CA −6.5 −17.3 −10.2 −26.2 CT/GA −7.8 −20.8 −7.6 −19.2 GA/CT−5.6 −13.5 −13.3 −35.5 CG/GC −11.9 −27.8 −8.0 −19.4 GC/CG −11.1 −26.7−14.2 −34.9 GG/CC −11.0 −26.6 −12.2 −29.7 Initiation 0.0 −10.8 0.0 −10.8

In general, these methods will result in estimates, not absolutelyaccurate values. Moreover, conditions like immobilization (see above),impurities, and nucleotide modification may all influence the actualT_(m) value of a given duplex or hybrid.

In some cases, an empirical approach must be used, while in others theempirical determination may be used to improve on the estimate derivedfrom the calculation. Generally, modifications like biotin, fluorescentdyes and the like will likely result in a lower T_(m), while somemodifications (like the backbone modification used in LNAs) may resultsin a higher T_(m). For LNA backbones, the nucleotides containing suchbackbones can, as an approximation, be assumed to be RNA nucleotides(resulting in more stable binding and hence higher T_(m) values).Therefore, an LNA-RNA hybrid could be calculated Like the correspondingRNA-RNA hybrid, while a nucleotide containing some LNA nucleotides andsome DNA nucleotides could be calculated on the basis of mixed energyvalues (for LNA nucleotides RNA values could be used, for DNAnucleotides DNA values are used).

For ease of use in molecular biology, the above methods are alsoimplemented on a number of servers for public use, such ashttp://biophysics.idtdna.com/ orhttp://www.biophp.org/minitools/melting_temperature/demo.php.

Thus, the person of ordinary skill is easily able to determine anestimate for the melting temperature for a given nucleotide sequence.For instance, for SEQ ID NO: 1, a T_(m) of between 50 and 54.4° C. couldbe obtained, while for SEQ ID NO: 3 (the miRNA 31 precursor sequence),between 67.4 and 76.2° C. could be obtained. These values are calculatedfor 15 mM monovalent cation (salt) concentration, which correspondsapproximately to 0.1×SSC buffer, that is, to stringent hybridizationconditions (see further above), with the proviso that the temperaturemust be adjusted accordingly (For the precursor, in this case the 65° C.temperature condition could be tested, while for the shorter maturemiRNA, the temperature must be lowered to about 45° C., depending onfurther factors like pH, divalent cations, target concentration and thelike. Correspondingly, an estimate for variations of a given sequencemay be obtained (providing that the target is matched in sequence).

The detection of a miRNA can be achieved by various methods. Forinstance, miRNA may be detected and quantified by deep sequencingmethods, relying upon the fact that more sequence reads are obtainedfrom miRNA molecules present more abundantly. Such methods are wellknown to the person of skill and described, inter alia, by Friedländer.,Nat Biotechnol (2008), 26(4): 407-415; Koh, BMC Genomics (2010), 10(11)Suppl 1: S6; Creighton, Brief Bioinform (2009), 10 (5): 490-497. miRNAabundance may further be determined by array technology. For instance,the Ncode human miRNA array available from Invitrogen, Carlsbad, Calif.,USA may be used to determine relative abundance of a large number ofhuman miRNAs in a sample. Chip arrays for detection and determination ofrelative abundance of miRNAs are also available from Affymetrix, SantaClara, Calif., USA, e.g., the U133 Plus 2.0. Affymetrix expression array(see. e.g., Ivanov, J Biol Chem (2010), 285: 22809-22817). Arrays basedupon LNA backbone probes (miRCURY LNA™ arrays) are, e.g., available fromExiqon, Vedbaek Denmark.

Further, the presence and relative abundance of a miRNA in a sample canbe assayed by in situ detection. Although the detection of microRNAs(miRNAs) in situ presents several technical challenges due to theirshort length, this can be overcome, e.g., by using digoxygenin-labeledlocked nucleic acid (LNA) oligonucleotide probes for detection (see,e.g., Sweetman, Methods Mol Biol (2011), 732: 1-8). Using this method,the subcellular distribution of miRNAs can be detected. In situ methodsare also useful for resolution of miRNA distribution on a tissue andorganism level (see, e.g., Diez-Roux, PLoS Biol (2011), 18; 9(1):e1000582). Further works by others have demonstrated the feasibility ofin situ hybridization and fluorescence-based detection for miRNAdetection and relative abundance determination (Lu J, Methods Mol Biol(2011), 680: 77-88; Mansfield, Methods. (2010), 52(4): 271-280; Gupta,Methods Mol Biol (2011), 676: 73-83; Debernardi, Methods Mol Biol(2010), 667: 33-45; Silahtaroglu, Methods Mol Biol (2010), 659: 165-171,also working with LNA nucleotides as probes; and Nuovo, Methods (2010),52(4): 307-315, demonstrating the method also for paraffin-embeddedsamples).

As mentioned, another example of a method of miRNA detection andquantification which may be employed in context of the diagnosis methodsprovided herein is quantitative PCR (qPCR). As the miRNA is a relativelyshort molecule, it is possible to extend its length by adding Adenosinemonomers to the strand (a technique known as polyadenylation) firstbefore reverse transcription and amplification. Briefly, the RNA may beextracted from a sample by a suitable reagent (e.g. Trizol reagentavailable from the above mentioned Invitrogen), poyadenylated in thepresence of ATP and poly(A) polymerase, reverse transcribed into DNAusing a poly(T) adapter and 5′ RACE sequence, and amplified using aforward primer derived from the 3′ end of the miRNA and a reverse RACEprimer (for details concerning primer design, RACE sequence etc. see.e.g., Shi, BioTechniques (2005), 39: 519-525. Improvements of thistechnique include designing the RACE primer with a nucleotide at its 3′end (constituting an A, C, or G, but not a T, so to exclude priminganywhere on the poly A sequence and enforce priming on the miRNAsequence; see also Reichenstein., J Virol Methods (2010), 163(2):323-328).

As an example, the following primers may be used for detection andquantification of miR-31 (SEQ ID NO: 1): Forward primer.5′-ACGCGGCAAGATGCTGGCA-3′ (SEQ ID NO: 29), Reverse primer.5′-CAGTGCTGGGTCCGAGTGA-3′ (SEQ ID NO: 30) (see Wang., Dis Markers(2009), 26(1): 27-34). Further examples for PCR quantification of miRNA31 include Liu, J Clin Invest (2010), 120(4): 1298-1309 (also detailingmicroarray assaying of miRNA31). Further, miRNA detection andquantitation using the Taqman system is readily available from AppliedBiosystems/Life technologies, Carlsbad, Calif., USA (see, e.g.,http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_(—)042142.pdf)as also described and exemplified herein in the appended Examples.Another example for quantitative PCR determination of mir31 uses astem-loop primer for reverse transciption and a forward and reverseprimers for PCR amplification of the miR-31 cDNA. Examples of suchprimers include for the stem-loop primer:gtcgtatccagtgcagggtccgaggtattcgcactggatacgacagctatgcctg (SEQ ID NO: 31);for the forward primer: tgaccgaggcaagatgc (SEQ ID NO: 32); and for thereverse primer: gtgcagggtccgaggt (SEQ ID NO: 33). Forward and reverseprimer overlap by one nucleotide on the miR-31 sequence, this however isnot enough to cause a primer dimer (for PCR conditions and furtherdetails see Ivanov, J Biol Chem (2010), 285: 22809-22817).

In another embodiment, the method for diagnosing bone disorders and/orcardiovascular disorders such as osteoporosis, osteopenia, bonefracture, impaired bone homeostasis, or cardiovascular diseases such asstroke, infarction, hypertension, thrombosis, vascular stenosis,coronary syndromes, vascular dementia, heart and renal failure oratherosclerosis in a subject, said method comprising the steps of

-   -   (a) contacting a biological sample from said subject with an        agent that binds to said polynucleotide being capable of        decreasing or suppressing expression of FZD3 and/or that binds        to miR-31 or its 5′ or 3′ isoforms or variants;    -   (b) detecting and evaluating the binding signal of the agent        of (a) with said polynucleotide and/or said miR-31 or its 5′ or        3′ isoforms or variants; and    -   (c) comparing the detected and evaluated the binding signal        of (b) with a correspondingly detected and evaluated binding        signal in a control sample,        wherein a stronger binding signal in the sample of the subject        compared to that of said control sample is indicative for a risk        of developing or having a bone disorder and/or cardiovascular        disorder.

In one embodiment of the diagnostic method provided herein, a method isprovided that is a method for diagnosing bone disorders and/orcardiovascular disorders in a subject, said method comprising the stepsof:

-   (a) detecting via a PCR method the expression level and/or quantity    of miR-31 (and isoforms and variants thereof as defined herein) in a    biological text sample; and-   (b) comparing the detected expression level and/or quantity of said    miR-31 (and isoforms and variants as defined herein) in said    biological sample with a corresponding expression level and/or    quantity of said miR-31 in a control sample.

Again, a control sample may be a sample derived from a disease-negative(or healthy) control patient. This would be a “negative control”.However, it is also envisaged, for example as a second or additionalcontrol, that such a control sample is a sample derived from a patientwho suffers from said bone disease and/or said cardiovascular disorder.This would be a positive control. The sample may be blood, blood serum,blood plasma or another biological fluid. As shown in the appendedexamples, plasma and serum are very useful. Methods for the assessmentof nucleic acid molecules, also of mino RNAs are well known in the art.Such methods comprise e.g. PCR, also and in particular quantitative PCRas well as real-time qPCR; see, e.g. Chen (2011) Methods Mol. Biol. 687,113-134, or Mestdagh (2008) Nuc. Acid Res. 36(21): e143.

In one embodiment (and as illustrated in the examples) the disorder tobe assessed or diagnosed in accordance with the invention is a bonedisorder, i.e. an osteopenia or osteoporosis and the like.

The invention also provides a method for the detection of apolynucleotide being capable of decreasing or suppressing expression ofFZD3 and/or which hybridizes to miR-31 or its 5′ or 3′ isoforms orvariants. A convenient method is based upon polymerase chain reaction(PCR), as this method is rapid, specific and sensitive. For PCR, aprimer is required that hybridizes specifically to the target nucleicacid. Moreover, a second primer is required to generate a PCR product byrepeated cycles of primer annealing, primer extension, and denaturing.The primer is chosen to maximize hybridization to the target nucleicacid while minimizing cross-hybridization to other nucleic acids presentin the sample. Examples of primer sequences and of calculating theoptimal hybridization temperature and other conditions for any chosenprimer sequence are given hereinbelow.

Accordingly, the invention provides a method for diagnosing bonedisorders and/or cardiovascular disorders in a subject, said methodcomprising the steps of:

-   -   (a) contacting a biological sample from said subject with a        first primer molecule which hybridizes to a polynucleotide being        capable of decreasing or suppressing expression of FZD3 and/or        which hybridizes to miR-31 or its 5′ or 3′ isoforms or variants,        forming a hybridization complex;    -   (b) contacting the hybridization complex of step (a) with a        second primer and a polymerase capable of extending the primer;    -   (c) repeatedly causing the primers to be extended by polymerase,        the product to denature, and the primers to hybridize to the        denatured product of polymerase extension,    -   (d) detecting and evaluating the product of step (c), resulting        in a value reflecting the amount of the polynucleotide being        capable of decreasing or suppressing expression of FZD3 and/or        which hybridizes to miR-31 or its 5′ or 3′ isoforms or variants        to which the first primer nucleic acid has hybridized;    -   (e) comparing the detected and evaluated value of (d) with a        correspondingly detected and evaluated value in a control        sample,    -   wherein a higher value resulting from the sample of the subject        compared to that of said control sample is indicative for a risk        of developing or having a bone disorder and/or cardiovascular        disorder.

Where the polynucleotide being capable of decreasing or suppressingexpression of FZD3 and/or which hybridizes to miR-31 or its 5′ or 3′isoforms or variants (the target nucleotide) is an RNA molecule, themethod preferably comprises the step of reverse transcription of thetarget nucleotide. Reverse transcription may be carried out by a primerthat overlaps a sufficient portion of the target molecule, preferably atits 3′ end. The length of the target molecule may be extended by thestep of polyadenylation prior to reverse transcription. In this case,the reverse transcription primer comprises a poly(t) sequence and atleast one nucleotide complementary to the target sequence. Preferably,said nucleotide is not a T. More preferably, the primer comprises thecomplement of two nucleotides of the target sequence, wherein the first(the penultimate nucleotide at the 3′ end of the primer) is not a T.

In another preferred embodiment, the reverse primer is a stem-loopprimer which comprises a short overlap with the (complement of the)target sequence and a stem-loop structure, as described and exemplifiedbelow.

In principle, the diagnosis methods described herein may employ a PCRtechnique (e.g., qPCR, RT-PCR, qRT-PCR, RT-qPCR or Light Cycler®) orother methods suitable to detect presence and/or amounts ofpolynucleotides. In the diagnosis methods of the present invention, thepresence and/or amount of a polynucleotide which is capable ofdecreasing or suppressing expression of FZD3 or a biologicallyderivative thereof and/or of miR-31 or its 3′ or 5′ isoforms or variantsis evaluated in a sample of a subject. PCR techniques and other methodssuitable for this purpose are known in the art and are also describedand exemplified herein. If the amount of said polynucleotide and/or ofmiR-31 or its 3′ or 5′ isoforms or variants is elevated compared to acontrol sample, the risk of developing or having a bone disorder and/orcardiovascular disorder is increased. For example, for the case ofemployment of a qPCR technique, total RNA of a subject's biologicalsample may be transcribed into cDNA. Then, specific primers hybridizingto said polynucleotide and/or of miR-31 or its 3′ or 5′ isoforms orvariants may be used for detection. The amount of polynucleotide and/orof miR-31 or its 3′ or 5′ isoforms or variants detected in the subject'ssample may then be compared to the amount of polynucleotide and/or ofmiR-31 or its 3′ or 5′ isoforms or variants of a control sample. Thehigher the amount is in the subject's sample compared to the controlsample, the higher is the risk of developing or having a bone disorderand/or cardiovascular disorder.

In context with the diagnosing methods described herein, a hybridizationor binding signal of the subject sample of (a) which is at least 50%,60%, 70% or 75% higher than that of the control sample may be indicativefor a risk of developing or having a bone disorder and/or cardiovasculardisorder such as osteoporosis, osteopenia, bone fracture, impaired bonehomeostasis, or cardiovascular diseases such as stroke, infarction,hypertension, thrombosis, vascular stenosis, coronary syndromes,vascular dementia, heart and renal failure or atherosclerosis. Thepolynucleotide capable of decreasing or suppressing expression of FZD3or a biologically active derivative thereof is preferably apolynucleotide to be inhibited in context of the present invention.Examples for biological samples in context of the present invention areblood, serum, plasma, other blood derived products, saliva, sperm fluid,vaginal fluid, urine, cerebrospinal fluid, or the like. In oneembodiment, the method is an in vitro method. In context of the presentinvention, the nucleic acid molecule hybridizing to the polynucleotideto be inhibited in context of the present invention or the agent, e.g.,an antibody, binding to the polynucleotide as described hereinabove mayfurther be conjugated to a marker or tagging molecule. Examples for suchmarker molecules for nucleic acids are fluorescent dyes excited andemitting at UV/VIS or infrared wavelengths like FITC, TRITC, Texas Red,Cy-dyes, alexa dyes (Bioprobes), etc. Examples for such marker/taggingmolecules for antibodies are enzymes like horse radish peroxidase,alkaline phosphatatase or fluorescent dyes excited and emitting atUV/VIS or infrared wavelengths like FITC, TRITC, Texas Red, Cy-dyes,alexa dyes, etc.

The appended sequence listing is part of the description.

The Figures show:

FIG. 1: Characterization of cells and SN used within this study.

-   -   (A) Pre-senescent (PD13) and senescent (PD53) endothelial cells        were stained for senescence associated β-galactosidase        activity. (B) Apoptotic cell death of HUVECs 48 h after        secretion into ASC or HUVEC medium was measured using Annexin        V-FITC and PI staining (N=3). (C) Representative microscope        pictures of two different ASC lines. (D) ASCs were stained for        expression of cell surface markers using flow cytometry.        Abbreviations: ASC, adipose-derived stem cell.

FIG. 2: Effects of HUVEC supernatants on proliferation anddifferentiation capacity of ASCs.

-   -   (A) ASCs were cultivated in the presence of conditioned medium        from pre-senescent (PD13) and senescent (PD53) cells and in        control medium. Viable cell numbers were analysed using a        hematocytometer and trypan blue staining. Error bars indicate        the standard deviations of 3 independent measurements. (B)        Treated cells were differentiated into the osteogenic lineage        detected using Alizarin Red staining. Representative pictures of        3 independent experiments are shown. Abbreviations: Undiff ctrl,        undifferentiated control; S, senescent; Y, young; C, control.

FIG. 3: Exosomes of senescent HUVECs increase proliferation and reducedifferentiation capacity of ASCs.

-   -   (A) Electron microscopy of HUVEC derived exosomes. The image        shows typical cup-shaped vesicles of ˜50 to 100 nm in        diameter. (B) Western blot analysis shows exosome marker protein        CD63. (C) Exosomes were labelled with anti-CD63 antibody and        visualized by electron microscopy. (D) ASCs were cultivated in        the presence of exosomes derived from pre-senescent (PD 13) and        senescent (PD53) cells and in control medium. Cell numbers were        detected using a hematocytometer and trypan blue staining. Error        bars indicate the standard deviations of 3 independent        measurements. (E) Treated cells were differentiated into the        osteogenic lineage. Ca deposits as marker for osteogenesis was        detected by Alizarin Red staining. Representative pictures of 3        independent experiments are shown. Abbreviations: Undiff ctrl,        undifferentiated control; S, senescent; Y, young; C, control.

FIG. 4: MiR-31 is increasedly secreted by senescent endothelial cells.

-   -   MiR-31 is upregulated in senescent HUVECs (A) as well as in        SN (B) and exosomes (C) derived from senescent HUVECs. (D)        Localization of miR-31 within exosomes by electron microscopy in        situ hybridization (EM-ISH). Abbreviations: S, senescent; Y,        young; SN, supernatant.

FIG. 5: ASCs take up exosomes derived from HUVECs.

-   -   (A) Intracellular levels of miR-31 in ASC after treatment with        SN or exosomes derived from pre-senescent and senescent        HUVECs. (B) Representative image of stable transfected senescent        endothelial cell expressing GFP. (C) Representative image of        ASCs after uptake of GFP-positive exosome 48 h after        incubation. (D) ASCs showing decreased levels of intracellular        miR-31 levels after transfection with the negative dominant        dynamin construct (K44A) and treatment with exosomes.        Abbreviations: SN, supernatant; S, senescent; Y, young; C,        control; WT, wild type.

FIG. 6: miR-31 alone reduces osteogenic differentiation of ASCs.

-   -   (A) Elevated miR-31 levels after transient transfection were        confirmed using TaqMan assay. (B) MiR-31 transfected cells were        differentiated into the osteogenic lineage (N=3).        Differentiation was detected using Alizarin Red staining.        Representative images of 3 independent experiments are shown.        Abbreviations: Undiff ctrl, undifferentiated control; ctrl,        control.

FIG. 7: FDZ3 is a target of miR-31 in ASCs.

-   -   (A) Significant upregulation of FDZ3 mRNA 4 days after        osteogenic differentiation start. (B) Significant downregulation        of FDZ3 mRNA in ASCs treated with senescent versus young        exosomes. (C) Downregulation of FDZ3 mRNA after transient        transfection of ASCs with 10 nM miR-31 precursor. Abbreviations:        OD, osteogenic differentiation; S, senescent; Y, young; C,        control; ctrl, control.

FIG. 8: Exosomes derived from plasma of elderly reduce osteogenicdifferentiation of ASCs.

-   -   (A) Electron microscopy of serum derived exosomes. The image        shows typical cup-shaped vesicles of ˜50 to 100 nm in        diameter. (B) Significant upregualtion of miR-31 levels in serum        samples derived form healthy old donors (N=27) compared to young        healthy controls (N=21). (C) ASCs were treated with exosomes        derived from old and young donors and differentiated into the        osteogenic lineage. Differentiation was detected using Alizarin        Red staining. Representative pictures of 2 independent        experiments are shown. (D) MiR-31 levels in plasma derived from        osteopenia patients were significantly increased compared to        healthy age matched controls.

FIG. 9: Proposed model summarizing the results.

-   -   Supernatant/exosomes derived from senescent endothelial cells        affect proliferation and differentiation potential of ASCs via        miRNA delivery. Thereby, the “senescent” environment hampers        tissue regeneration and promotes cell growth which is one of the        risk factors in the development of cancer.

FIG. 10: MiR-31 is also induced by H₂O₂ in exosomes derived fromsenescent versus untreated HUVECs.

-   -   Exosomes were harvested from H₂O₂ pulsed endothelial cells after        14 days, when no additional cell proliferation was observed in        the cells treated with 75 μM tBHP. In contrast, after exposure        to 35 μM tBHP the cells completely recovered and resumed growth.

FIG. 11: Levels of osteogenic differentiation of ASCs after transfectionwith miR-31, anti-miR-31, non-targeting control miRNAs andnon-transfected cells.

-   -   While miR-31 significantly inhibits osteogenesis as analysed by        Alizarin red staining, anti-miR-31 significantly increases        osteogenesis.

FIG. 12: Plasma levels of miR-31.

-   -   Plasma levels of miR-31 of 10 male patients diagnosed with        osteopenia were compared to age matched healthy controls. 7 out        of 10 patients show higher levels of miR-31 normalized to U6B        snRNA, resulting in a highly significant difference as        calculated by Student's t testing.

The Examples illustrate the invention.

EXAMPLE 1 Cell culture Human Umbilical Vein Endothelial Cell (HUVEC)

Endothelial cells were isolated from human umbilical veins as described(Chang, Exp Cell Res (2005), 309: 121-136; Jaffe, J Clin Invest (1973),52: 2745-2756). HUVECs were cultivated in gelatin precoated flasks inM199 with Earle's salts supplemented with 4 mM glutamine, 15% fetal calfserum (FCS) and 10% endothelial cell growth supplement (ECGS) containing170 U/ml heparin at 37° C. in a humidified atmosphere with 5% CO₂. Cellswere passaged once or twice a week at a split ratio of 1:2 to 1:4according to the growth rate. HUVECs were cultivated to senescence andstained for senescence associated 3-galactosidase (SA-β-gal) activity asdescribed previously (Chang, loc cit). For collection of supernatants,contact inhibited (quiescent, PD19) and senescent (PD53/95% SA-β-galpositive) cells were allowed to secrete into ASC or HUVEC medium,depending on the experiment, for 48 h. Then supernatants were collected,centrifuged at 1900 g, 4° C. and used freshly for exosome preparation orstored at −80° C. Supernatant volumes were normalized to the number ofsecreting HUVECs in one flask at the time of supernatant harvest. Cellculture medium incubated for 48 h at 37° C. was used as additionalcontrol.

Human Adipose-Derived Stem Cells (ASCs)

Subcutaneous adipose tissue was obtained during outpatient tumescenceliposuction under local anestesia. ASCs were isolated as describedbefore (Wolbank, Tissue Eng (2007), 13: 1173-1183; Wolbank, Tissue EngPart A (2009)) and cultured in DMEM-low glucose/HAM's F-12 supplementedwith 4 mM L-glutamine, 10% fetal calf serum (FCS, PAA) and 1 ng/mLrecombinant human basic fibroblast growth factor (rhFGF, R&D Systems) at37° C., 5% CO₂ and 95% air humidity. Cells were passaged once or twice aweek at a split ratio of 1:2 according to the growth rate. ASCs werecharacterized due to the expression of specific surface markers (CD13,CD14, CD34, CD45, CD73, CD90, HLA ABC, HLA DR) by flow cytometry (FACSCalibur, Beckton Dickenson) using standard procedures.

Differentiation into Osteogenic Lineage

All differentiation protocols were carried out in 24 well cell cultureplates. For osteogenic differentiation, ASCs were seeded at a density of2×10³ cell per well. 72 h after seeding cells were incubated withosteogenic differentiation medium (DMEM-low glucose, 10% FCS, 4 mML-glutamine, 10 nM dexamethasone, 150 μM ascorbate-2-phosphat, 10 mM(3-glycerolphosphate and 10 nM vitamine-D3) up to 4 weeks.

Alizarin Red Staining

For Alizarin Red staining of calcified structures, cells were fixed for1 h in 70% ethanol at −20° C. After brief rinsing, cells were stainedfor 20 min with 40 mM Alizarin Red solution (Sigma) and washed with PBS.For quantification, Alizarin Red was extracted for 30 min using 200 μl0.1 M HCl/0.5% SDS solution. The extracted dye was measured at 425 nm.

EXAMPLE 2 Transfections

ASCs were transfected using siPORT™ NeoFX™ transfection reagent (AppliedBiosystems). Cells were transfected with 10 nM Precursor hsa-miR-31, 100nM anti-miR-31 or 10 nM negative control 2# (Ambion) according to themanufacturer's protocol. Cells were harvested after 24 or 48 h ordifferentiation was started as described before three days aftertransfection.

Moreover, ASCs were transfected with a 0.5 μg dominant negative dynaminconstruct (K44A) or dynamin wild type construct (provided by Mark A.McNiven Department of Biochemistry and Molecular Biology & Center forBasic Research in Digestive Diseases, Mayo Clinic and Graduate School,Rochester, Minn. 55905, USA) using Metafectene Pro (Biontex LaboratoriesGmbH) according to manufactures protocol.

EXAMPLE 3 Assessment of Apoptotic Cell Death

HUVECs were seeded in 12-well cell culture plates and were allowed tosecrete into ASC medium for 48 h. Thereafter, the cells were detachedusing 50 mM EDTA and stained with Annexin V-FITC and PI (Roche)according to the manufacturer's instructions. Analysis of the percentageof apoptotic and necrotic/late-apoptotic cells were performed using aFACS-Calibur and the CellQuest software (Becton Dickinson).

EXAMPLE 4 Quantitative Real-Time PCR

Alizarin red stainings were confirmed using different osteogenicdifferentiation marker genes. Therefore, total ASCs RNA was isolatedusing Trizol (Invitrogen) at different time points before and duringosteogenesis. Reverse transcription was performed using DyNAmo cDNASynthesis Kit (Biozym) and qPCR was performed using the RotorGene2000(Corbett). For miRNA analysis, specific TaqMan assays (AppliedBiosystems) were used according to manufactures protocol.

For isolation of RNA from blood samples, 250-500 μl serum was used toisolate total RNA using Trizol LS reagent (Invitrogen). To allow fornormalization of sample-to-sample variation in RNA isolation, 25 fmolsynthetic C-elegans miRNAs cel-miR-39 were added before isolation. Serumsamples (27 healthy old donors and 21 healthy young donors) wereobtained from R. Westendorp, Department of Gerontology & GeriatricsC2-R, Leiden University Medical Center, The Netherlands. Moreover 4osteopenia serum samples obtained from P. Pietschmann, Department ofPathophysiology, General hospital, Vienna. Institutional ethicscommittees approved the study, and written, informed consent has beenobtained from each subject.

EXAMPLE 5 Lentiviral Transduction

Senenscent HUVECs were transduced with a lentiviral vector containingGFP protein kindly provided by Pidder Jansen-Durr, Institute forBiomedical Aging Research, Innsbruck, Austria (Muck, Rejuvenation Res(2008), 11: 449-453). Briefly, lentiviral transduction was performedusing senescent and pre-senescent HUVECs. Generally, 100000-150000senescent and 50000 pre-senescent HUVECs were plated in a 6-well plateand incubated over night. A multiplicity of infection (MOI) of two toeight with 8 μg/ml Polybrene, which increases the infection efficiency,was used to produce stably transduced cells. As selection pressure, 10μg/ml Blasticidin was added to the medium. After 6-8 days, clones werestable and contamination with Lentiviral particles was tested byincubation of HeLa's with SN for 4 days. Thereafter, SN was harvestedand used for exosomes purification as described before.

EXAMPLE 6 Exosome Purification

Exosomes were purified by filtration and differential centrifugation asdescribed previously (Lehmann, Cancer Res (2008), 68: 7864-7871). Inbrief, supernatants were collected after incubation of 48 h. Thisconditioned medium will be centrifuged at 500 g for 10 min to sedimentcells and at 14000 g for 15 min to eliminate cell debris and filteredthrough a 0.22 μm filter excluding a fraction of apoptotic bodies.Exosomes are then sedimented by ultracentifugation at 100000 g for 60min and the resultant pellet is washed with PBS. Exosomes will be usedas fresh preparations for electron microscopy or conserved at −80° C.for further analysis. For differentiation studies, exosomes derived from2×10⁴ HUVECs in 50 μl PBS were added per well ASCs.

EXAMPLE 7 Electron Microscopy

Purified exosomes were left to settle on nickel coverslips (200 mesh,hexagonal, Pioloform-coated Athene copper grids) After fixation with 4%paraformaldehyd, exosomes were stained with 2% uranyl acetate for 30sec, coverslips were left to dry and visualized using a transmissionelectron microscopy (TEM), Philips model CM 12 electron microscope(Philips, Eindhoven, NL).

For electron microscopy, in-situ hybridization (EM-ISH) exosome pelletswere permeabilized with 0.1% Triton-X for 5 min at room temperature.After washing with PBS, exosomes were incubated for at least 4 h withhybridization buffer as described previously (Obernosterer, Nat Protoc(2007), 2: 1508-1514). For each sample, 1 pM of the LNA DIG-labelledsingle stranded probe (Exiqon, Denmark) was denaturated in denaturizinghybridization buffer (containing 50% formamide, 5×SSC, 5×Denhardt'ssolution, 0.1% Tween, 0.25% CHAPS, 200 μg/ml yeast RNA, 500 μg/ml salmonsperm DNA) by incubation at 80° C. for 5 min. Probes were placed on icequickly. Exosomes were mixed with the probe and hybridized at 50° C.over night. After hybridization, samples were washed stringently with0.2×SSC at 60° C. for 1 h. Thereafter, exosomes were incubated withAnti-DIG antibody (Roche) for 30 min and an additional hour with thesecond 5 nm gold particle labelled antibody (Sigma). After washing withPBS, exosomes were embedded in Epon, sections on average, approximately80 nm were cut using a Ultramicrotom and were then analysed usingtransmission electron microscopy (TEM), Philips model CM 12 electronmicroscope (Philips, Eindhoven, NL).

EXAMPLE 8 Western Blot

Total proteins were extracted and separated on polyacrylamide gelsbefore transfer to a PVDF membrane (Roth, Germany). The membrane wasblocked in 5% skimmed milk, incubated with the CD63 antibody (H-193,Santa Cruz) followed by horseradish peroxidase-coupled secondaryantibody and subjected to enhanced chemiluminescence using ECL WesternBlotting Substrate (Pierce) on a Chemidoc (Biorad).

EXAMPLE 9 Effects of HUVEC Supernatants on Proliferation andDifferentiation Apacity of ASCs

In order to test if senescent endothelial cells might contribute to thestem cell inhibiting systemic environment (Conboy, Nature (2005), 433:760-764), well characterized pre-senescent and senescent HUVECs (PDLbetween PD13 and PD53) were used as established earlier (Chang, Exp CellRes (2005), 309: 121-136). Senescent cells showed a large flattenedmorphology and stained positive for SA-β-gal activity (FIG. 1A). Inorder to produce endothelially secreted factors in ASC medium,endothelial cells were exposed to ASC medium for 48 h. In order toexclude that excessive cell death influences the “secretome”, the basallevel of cell death of endothelial cells during the time of “harvesting”using Annexin V and PI staining was tested. Their was no significantdifference visible between pre-senescent and senescent HUVECs (FIG. 1B).

Furthermore, ASCs showing typical morphology from 6 different donors(examples FIG. 1C) were used and their identity was confirmed bycharacterization of their surface marker profile (FIG. 1D) as describedearlier (Wolbank, 2009, loc cit; Yañez, Stem Cells (2006), 24:2582-2591).

In order to test and compare the influence of conditioned medium frompre-senescent and senescent HUVECs on growth characteristics of ASCs,cells were incubated with the respective supernatant for a period of 5d. Although cell viabilities remained unaltered in each test settingthroughout the whole experiment (data not shown), ASCs cultivated in thepresence of senescent supernatants reached significantly higher cellnumbers than cells cultivated in the presence of pre-senescent orcontrol medium (FIG. 2A). Additionally, supernatants derived fromsenescent cells significantly reduced the osteogenic differentiationpotential as stained by Alizarin Red of ASCs after 21 d of incubationwith differentiation medium (FIG. 2B).

EXAMPLE 10 Exosomes of Senescent HUVECs Increase Proliferation andReduce Differentiation Capacity of ASCs

Exosomes are membrane coated vesicles, which are between 40 and 100 nmin diameter, and originate from intracellular multivesicular bodies(Pap, Inflamm Res (2009), 58: 1-8). Since recently exosomes have beendescribed as paracrine signalling molecules in various settings(Deregibus, Blood (2007), 110: 2440-2448; Hunter, PLoS ONE (2008), 3:e3694; Lehmann, loc cit; Pap, loc cit), it was tested whether exosomesmight contribute to the changes in ASC behaviour. Therefore, exosomeswere isolated from HUVEC supernatants by sequential centrifugation steps(Deregibus, loc cit). To confirm the identity of the exosomes electronmicroscopy was performed. The size distribution strongly indicates thatno apoptotic vesicles are visible in our exosome preparations sinceapoptotic bodies are >500 nm in size (Reich, Exp Cell Res (2009), 315:760-768) (FIG. 3A). Furthermore, Western blot analysis (FIG. 3B) as wellas immunogold labelling (FIG. 3C) using antibodies against CD63 surfaceprotein—a commonly used marker of exosomes (Valadi, Nat Cell Biol(2007), 9: 654-9). (FIG. 3A-C) suggest that the exosome isolation wassuccessful.

When ASCs were treated with exosomes derived from senescent HUVECs, theproliferation rate was again significantly increased (FIG. 3D) comparedto stem cells treated with exosomes derived from pre-senescentendothelial cells. Moreover, osteogenic differentiation capacity wassignificantly decreased by ˜50% when cells were treated with senescentexosomes (FIG. 3E), whereas adipogenic differentiation was notinfluenced (data not shown).

EXAMPLE 11 miR-31 is Secreted by Endothelial Cells

It was shown that beside different proteins, also miRNAs are packed intoexosomes (Valadi, loc cit). miR-31 was shown to be upregulated insenescent HUVECs (FIG. 4A). Furthermore, it was tested whether it isalso present in HUVEC culture supernatants (FIG. 4B) and exosomesderived from senescent versus pre-senescent endothelial cells (FIG. 4C).Since up to 12-fold changes were detected by qPCR, the localization ofmiRNAs within exosomes was confirmed by electron microscopy in situhybridization (EM-ISH). While so far only biochemical assays suggestedthat miRNAs are inside of exosomes, in context with the presentinvention, first microscopic proof is provided that miRNAs are indeedpackaged into exosomes (FIG. 4D).

EXAMPLE 12 ASCs Take Up Exosomes Derived from HUVECs

In order to test whether endothelial derived miR-31 is taken up by ASCs,ASCs were incubated with SN and exosomes from HUVECs. Indeed, after 48 ha 4-5 fold increase of miR-31 inside of ASCs was observed (FIG. 5A).From these data it was not clear if exosomes are taken up by ASCs or ifother components contained within the exosome preparations would inducede novo transcription of miR-31 within ASCs. Therefore, stabletransfected senescent endothelial cells expressing GFP (FIG. 5B) wereprepared as it was recently published that GFP is packaged into exosomes(Deregibus, loc cit). Two days after incubation with GFP positiveexosomes, ASCs showed GFP signals in a punctuate pattern within thecytoplasm while ASCs treated with the supernatant depleted ofGFP-exosomes showed no signals (FIG. 5C). To further confirm the exosomeuptake, ASCs were transfected with a dominant negative dynamin construct(K44A) and dynamin wild type construct as control (Cao, Mol Biol Cell(1998), 9: 2595-2609; Cao, J Cell Sci (2000), 113 (Pt 11): 1993-2002).Dynamin was shown to be responsible for endocytosis in eukaryotic cells(Cao, 1998, loc cit; Cao, 2000, loc cit) and endocytosis is responsiblefor uptake of exosomes (Valadi, loc cit). Indeed, as shown in FIG. 5D,miR-31 levels were decreased in ASCs transfected with the dominantnegative dynamin construct (K44A).

These results show that miR-31 is indeed taken up via the exosomes andnot by induction in consequence of signal transduction through themembrane, it seems that this uptake resembles transfections in vitro,where lipids containing DNA or RNA deliver their cargo into the cells.

EXAMPLE 13 miR-31 Alone Reduces Osteogenic Differentiation of ASCs

In order to investigate the effect of miR-31 alone on differentiation,ASCs were transiently transfected with miR-31. Elevated miR-31 levelswere confirmed using TaqMan assay (FIG. 6A). The influence of miR-31 onthe differentiation capacity of ASCs was invesitigated and osteogenicdifferentiation was significantly inhibited by around 2-fold when ASCswere transiently transfected with miR31 (FIG. 6B), a similar range ofinhibition as seen with the exosomes alone. These data indicate thatmiR-31 is an inhibitor of osteogenic differentiation that might besecreted by endothelial cells especially at senescence.

EXAMPLE 14 Target of miR-31

FZD3 mRNA levels were indicated to be increased in MSCs under osteogenicconditions (Baksh, J Cell Biochem (2007), 101: 1109-1124). In context ofthe present invention, it was upregulated after 4 days compared to cellstreated with control medium (FIG. 7A). Moreover, FZD3 levels weresignificantly downregulated after treatment with senescent exosomescompared to treatment with young exosomes and control treated cells(FIG. 7B). 24 h after miR-31 transfection, FDZ3 was also downregulatedbut did not reach significant levels, which might be explained due tothe very low mRNA levels of FDZ3 when cells are not differentiating(FIG. 7C). Thus, FZD3 represents not only a marker but also a necessaryfactor for osteogenic differentiation and might be a direct target ofmiR-31 also in ASCs.

EXAMPLE 15 Effects of Exosomes Derived from Plasma on ASCs

It was confirmed that exosomes existed in the blood plasma usingelectron microscopy (FIG. 8A). Then, RNA was isolated from blood serumfrom 21 healthy young (19-47 years) and 27 old donors (50-91 years) andmiR-31 levels were analyzed. As has been found in context of the presentinvention, they were significantly increased in elderly people andshowed larger variations compared to young donors (FIG. 8B).Furthermore, cells were treated with exosomes derived from 1 ml plasma/1ml medium for 72 h, afterwards differentiation was started. Using 4different donors, a significant decrease of around 3-fold in osteogenicdifferentiation of ASCs treated with “old” exosomes (FIG. 8C) was found.The very low differentiation in control samples could be explained bythe extremely fast differentiation of the cells treated with exosomesderived from serum. Additionally, herein it was shown that miR-31 levelsin plasma derived from osteopenia patients were elevated compared tohealthy age matched controls (FIG. 8D).

EXAMPLE 16 Oxidative Stress Induces Senescence

Oxidative stress is known to be associated with endothelial senescenceand dysfunction (Seals, Clin Sci (Lond) (2011), 120(9): 357-375). Totest whether oxidative stress induces secretion of miR-31, SIPS(stress-induce premature senescence) was induced by tBHP treatment ofHUVECs on 5 consecutive days for 1 h each by adding tBHP to a finalconcentration of 75 μM, 50 μM or 35 μM in the medium. Permanent growtharrest was induced by 75 μM tBHP as assessed by microscopic follow upfor 14 d according to the protocols described in Unterluggauer, ExpGerontol (2003), 38: 1149-1160.

As a result, miR-31 was found to be elevated in exosomes ofstress-induced premature senescent endothelial cells. Using increasingdoses of H₂O₂ (35 μM, 50 μM and 75 μM tButylhydroperoxide (tBHP)), up to5-fold induction of miR-31 was observed in stress induced senescentHUVECs versus unstressed controls, similar to the levels inreplicatively senescent HUVECs (FIG. 10).

Furthermore, the increase of miR-31 in the supernatant of senescentendothelial cells is not restricted to HUVECs that are derived fromhuman umbilical vein endothelial cells, but was also found in senescenceof human retinal endothelial cells as well as in senescent human liverderived endothelial cells versus early passage control cells (data notshown).

EXAMPLE 17 miR-31 Antagonist Inhibits Osteogenic Differentiation

In order to test whether miR-31 inhibition improves osteogenicdifferentiation, antagonistic locked nucleic acids (LNA) against miR-31were analyzed (Ambion, product ID: AM11465; P/N AM17000). Experimentalprocedures for transfection of ASCs were performed analogously as donein Example 2 above.

As has been found, a significant increase in Ca-deposition as analysedby Alizarin red staining was observed, while transient increase inmiR-31 resulted in decreased osteogenic differentiation (FIG. 11). Thesedata show that inhibition or removal of miR-31 systemically or locallywill improve osteoblast formation, e.g., in osteoporosis and afterfractures.

EXAMPLE 18 miR-31 Inhibits Osteogenesis in Mouse Model System

In order to confirm that miR-31 is a general regulator of osteogenesisnot only in the human system, C3Ht101/2 cell line was used, a mousemesenchymal mulitpotent cell line that can be induced to undergoosteogenic differentiation by addition of BMP2 (Richard, PLoS Genetic(2005), 1(6): e74). As readout, cells were co-transfected with areporter construct encoding luciferase under control of the osteocalcinpromoter (Feichtinger, Tissue Eng Part C Methods (2010), 17(4):401-410).

The influence of miRNA-31 transfection on osteogenic differentiation wasfurthermore analyzed using the (C2C12) C3Ht10 1/2 cell line inconjunction with an osteocalcin specific reporter gene assay(Feichtinger, Tissue Eng Part C Methods (2010), 17(4): 401-410). (C2C12)C3Ht10 1/2 cells are capable of differentiating to the osteogeniclineage upon treatment with recombinant BMPs, which is observable by theinduction of alkaline phosphatase, osteocalcin and other osteoblastspecific genes. The cells, seeded in a T175 flask, were firsttransfected with 99 μg of the osteocalcin reporter system and then 24 hpost reverse transfected with 30 nM miRNA-31 or a scrambled miRNA ctrl#12 as control using Ambions siPORT NeoFX. Osteogenic differentiationwas induced using 300 ng/ml recombinant BMP2 (InductOS, Pfizer),controls were not induced with growth factor. Osteocalcin reporteractivity was assessed after 6 d of differentiation by determiningmetridia luciferase activity in the cell culture supernatants usingClontech Ready-To-Glow Secreted Luciferase System Kit.

As a result, inhibition of osteogenesis by transient miR-31overexpression was confirmed also in the mouse model. Furthermore,similar to young versus old healthy humans, it was found that the serumof old mice contains 3-10 fold higher concentrations of miR-31 than theserum of isogenic young mice as controls (data not shown).

EXAMPLE 19 Analysis of miR-31 in Plasma of Osteopenia Patients

Plasma of 10 male patients diagnosed with osteopenia were compared toage matched controls provided by P. Pietschmann, Department ofPathophysiology, General hospital, Vienna. Institutional ethicscommittees approved the study, and written, informed consent has beenobtained from each subject.

Plasma was prepared according to standard procedures clinical proceduresas recommended for the analysis of miRNAs (Taylor, Methods Mol Biol(2011), 728: 235-246; Kroh, Methods (2010), 50: 298-301). Total RNA wasisolated using Trizol LS. The qPCR was then performed using Taqman®protocol, where in a first step a specific reverse transcription andthen amplification is performed. The amplification of specific miR-31amplicons is monitored by displacing a fluorescently labelled probe bythe amplification. The assays were run in triplicates and differentialexpression of miR-31 was normalized to the small U6 snRNA. In addition aspike in control of a C. elegans specific miRNA was performed tonormalize for miRNA recovery during the full RNAisolation—quantification procedure. PCR was performed as detailed below.

TaqMan Protokoll (hsa-miR-31) Applied Biosystems

cDNA Synthesis:

2 x mastermix: containing miR-31 and U6 primers volume [μl]: # ofreactions: 100 mM dNTPs 0.10 MultiScribe RT 0.60 10 x Buffer 1.00 RNaseinhibitor 0.12 Nuclease free water + RNA 5.18 RT-Primer 2.00 Total 9.00RNA (10 ng/μl) 1.00 Total 10.00

Program:

-   16° C.: 30 min-   42° C.: 60 min-   85° C.: 5 min-   4° C.: pause

TaqMan Primer: Applied Biosystems

Assay Name hsa-miR-31 Part Number 4427975 AB Assay ID 001100 Assay TypeMature miRNA Availability Inventoried

Mature MicroRNA Details Mature miRNA GGCAAGAUGCUGGCAUAGCUG (SEQ ID NO:Sequence 19) Target Species Gorilla gorilla, Macaca mulatta,Macaca nemestrina, Pan paniscus, Pongo pygmaeus, Pan troglodytesmiRBase ID ggo-miR-31, mml-miR-31, mne-miR-31, ppa-miR-31,ppy-miR-31, ptr-miR-31 miRBase MIMAT0002381, MIMAT0002379, AccessionMIMAT0002383, MIMAT0002384, Number MIMAT0002382, MIMAT0002380miRBase Alias hsa-miR-31 v9.2 Gene Family ID MIPF0000064, mir-31

Device and Software:

-   Rotor-Gene 6000; Rotor-Gene Software 6000, Series Software 1.7

What is claimed is:
 1. A method for diagnosing bone or cardiovasculardisorders in a subject, which method comprises: (a) contacting abiological sample from said subject with a nucleic acid molecule whichhybridizes to miR-31 or its 5′ or 3′ isoforms or variants, or contactingsaid biological sample with an agent that binds to miR-31 or its 5′ or3′ isoforms or variants; (b) detecting and evaluating the hybridizationsignal of the nucleic acid molecule of (a) or detecting and evaluatingthe binding signal of the agent of (a) with said polynucleotide and/orsaid miR-31 or its 5′ or 3′ isoforms or variants; and (c) comparing thedetected and evaluated hybridization signal of (b) or comparing thedetected and evaluated the binding signal of (b) with a correspondinglydetected and evaluated hybridization or binding signal in a controlsample, wherein a stronger hybridization signal or a stronger bindingsignal in the sample of the subject compared to that of said controlsample is indicative for a risk of developing or having a bone orcardiovascular disorder.
 2. The method according to claim 1, whereinsaid hybidizing nucleic acid molecule or said binding agent isconjugated with a marker molecule and/or a tagging molecule.
 3. Themethod according to claim 1, wherein said agent to be employed is aspecific protein or protein fragment.
 4. The method according to claim3, wherein said specific protein or protein fragment is an antibody or afragment thereof or is a modified transcription factor capable ofbinding to and/or interacting with polynucleotide that is capable ofbinding to and/or interacting with miR-31 or its 5′ or 3′ isoforms orvariants.
 5. The method according to claim 1 wherein said bone orcardiovascular disorder is osteoporosis, osteopenia, bone fracture,impaired bone homeostasis, or cardiovascular diseases such as stroke,infarction, hypertension, thrombosis, vascular stenosis, coronarysyndromes, vascular dementia, heart and renal failure oratherosclerosis.
 6. A method for diagnosing bone or cardiovasculardisorders in a subject, which method comprises: (a) detecting via a PGRmethod the expression level and/or quantity of miR-31 or of an isoformor variant thereof in a biological text sample; and (b) comparing thedetected expression level and/or quantity of said miR-31 or of saidisoform or variants in said biological sample with a correspondingexpression level and/or quantity of said miR-31 in a control sample. 7.The method according to claim 6 wherein the expression level and/orquantity of miR-31 or of an isoform or variant thereof is obtained bycontacting a biological sample from said subject with a nucleic acidmolecule which hybridizes to miR-31 or its 5′ or 3′ isoforms orvariants.
 8. The method according to claim 7 wherein the nucleic acidmolecule is at least one primer.
 9. The method according to claim 8wherein one primer is a forward primer that is complementary to asequence of the 5′-end of the polynucleotide to be detected, whileanother primer is a reverse primer that is complementary to a sequenceof the 3′-end of the polynucleotide to be detected.
 10. The methodaccording to claim 9 wherein each primer has a length of 12 to 30nucleotides and is not necessarily 100% complementary to the respectivesequence of the polynucleotide to be detected as long as the primer canhybridize to the polynucleotide.
 11. The method according to claim 9wherein the reverse primer is a stem-loop primer which comprises a shortoverlap with the complement of the target sequence and a stem-loopstructure.
 12. The method according to claim 9 wherein the primers areconjugated to marker or tagging molecules.
 13. The method according toclaim 1 wherein said bone or cardiovascular disorder is osteoporosis,osteopenia, bone fracture, impaired bone homeostasis, or cardiovasculardiseases such as stroke, infarction, hypertension, thrombosis, vascularstenosis, coronary syndromes, vascular dementia, heart and renal failureor atherosclerosis.